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EP2280736B1 - Zusammensetzungen und verfahren zur herstellung von emulsionen für kernmagnetresonanzverfahren und andere anwendungen - Google Patents

Zusammensetzungen und verfahren zur herstellung von emulsionen für kernmagnetresonanzverfahren und andere anwendungen Download PDF

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Publication number
EP2280736B1
EP2280736B1 EP09739238.5A EP09739238A EP2280736B1 EP 2280736 B1 EP2280736 B1 EP 2280736B1 EP 09739238 A EP09739238 A EP 09739238A EP 2280736 B1 EP2280736 B1 EP 2280736B1
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Prior art keywords
cells
cell
emulsion
labeled
tissue
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English (en)
French (fr)
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EP2280736A2 (de
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Eric T. Ahrens
Jelena Janjic
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Celsense Inc
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Celsense Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
    • A61K49/0078—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion microemulsion, nanoemulsion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1896—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes not provided for elsewhere, e.g. cells, viruses, ghosts, red blood cells, virus capsides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Cellular therapy is broadly defined as the treatment of human disease by the administration of therapeutic cells that have been selected, multiplied, and pharmacologically treated outside the body, or ex vivo. These cells may be derived from the patient (autologous cells), from another human (allogenic cells), from other organisms (xenogenic cells), or from immortalized cell lines.
  • Cells represent the ultimate therapeutic system because of their ability to carry out complex functions and their responsiveness to changes in the surrounding tissue or host organism.
  • cells can be isolated, grown in quantity ex vivo, and implanted in patients to produce and secrete soluble factors that directly address the mechanism of disease.
  • Cells can also accomplish tasks as complex as reconstitution of tissues, organs, or immune responses based on their ability to home to specific sites within the body, to exit from circulation, and to integrate into specific tissue or differentiate into new tissue.
  • Other cellular therapeutics can be programmed for tumor killing or treating metastases (e.g., immunotherapeutics).
  • PET Positron emission tomography
  • Magnetic resonance imaging is a widely used clinical diagnostic tool because it is non-invasive, allows views into optically opaque subjects, and provides contrast among soft tissues at reasonably high spatial resolution.
  • Conventional MRI focuses almost exclusively on visualizing anatomy and has no specificity for any particular cell type.
  • the 'probe' used by conventional MRI is the ubiquitous proton ( 1 H) in mobile water molecules. New classes of exogenous MRI probes or reagents are needed to facilitate cell-specific imaging in living subjects.
  • the application discloses an aqueous composition as characterized in the appended claims said aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, an emulsifier, a surfactant co-mixture, and an additive, wherein the emulsifier comprises glycerol polyethylene glycol ricinoleate.
  • the surfactant co-mixture comprises 70 mol % lecithin, 28 mol % cholesterol, and 2 mol % DPPE.
  • the additive is propylene glycol.
  • the composition comprises perfluor-15-crown-5 ether or PFPE oxide in the range of 20% to 50% w/v. In certain embodiments, the composition comprises perfluor-15-crown-5 ether or PFPE oxide in the range of 25% to 35% w/v. In certain embodiments, the composition comprises perfluor-15-crown-5 ether or PFPE oxide in the range of 30% to 40% w/v. In certain embodiments, the composition comprises perfluor-15-crown-5 ether or PFPE oxide in the range of 35% to 36% w/v. In certain embodiments, the composition comprises perfluor-15-crown-5 ether or PFPE oxide in 35.6% w/v.
  • the composition comprises the emulsifier in the range of 1% to 10% w/v. In certain embodiments, the composition comprises the emulsifier in the range of 1% to 5% w/v. In certain embodiments, the composition comprises the emulsifier in 3% w/v.
  • the composition comprises propylene glycol in the range of 1 % to 10% w/v. In certain embodiments, the composition comprises propylene glycol in the range of 1% to 5% w/v. In certain embodiments, the composition comprises propylene glycol in 2% w/v. In certain embodiments, the composition comprises the surfactant co-mixture comprising lecithin, cholesterol, and DPPE in the range of 1% to 10% w/v. In certain embodiments, the composition comprises the surfactant co-mixture comprising lecithin, cholesterol, and DPPE in the range of 1% to 5% w/v. In certain embodiments, the composition comprises the surfactant co-mixture comprising lecithin, cholesterol, and DPPE in 2% w/v.
  • the application discloses an aqueous composition as characterized in the appended claims said aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide in 35.6% w/v, an emulsifier in 3.0% w/v, a surfactant co-mixture in 2.0% w/v, wherein the surfactant co-mixture comprises lecithin, cholesterol, and DPPE, and an additive in 2.0% w/v, wherein the additive is propylene glycol and wherein the emulsifier comprises glycerol polyethylene glycol ricinoleate.
  • an aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and a block copolymer, wherein the composition comprises perfluoro-15-crown-5 ether or PFPE oxide in the range of 10% to 20% w/w, and wherein the composition comprises the block copolymer in the range of 0.1% to 2.0% w/w.
  • the block copolymer may be a tri-block copolymer which comprises polyethyleneoxide and polypropyleneoxide.
  • the block copolymer may be poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) tri-block copolymer comprising 80% PEO content.
  • the composition may comprise perfluoro-15-crown-5 ether or PFPE oxide in the range of 12% to 17% w/w or in 15% w/w. Moreover, it is described herein that the composition may comprise the block copolymer in the range of 0.1% to 1.0% w/w or in 0.6% w/w.
  • aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide in 15% w/w and a block copolymer in 0.6% w/w is also described herein.
  • the block copolymer may be a tri-block copolymer which comprises polyethyleneoxide and polypropyleneoxide.
  • the block copolymer may be poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) tri-block copolymer comprising 80% PEO content.
  • composition may further comprise protamine sulfate in the range of 0.01% to 1.0% w/w, in the range of 0.01% to 0.5% w/w, in the range of 0.01 % to 0.1% w/w or in 0.04% w/w.
  • the application discloses an emulsion comprising a composition of any one of the embodiments described herein.
  • the emulsion has a mean droplet size of less than 200 nM in diameter.
  • the emulsion is stable at temperatures ranging from 4 °C to 37 °C.
  • the emulsion has a polydispersity index ranging from 0.1 to 0.2.
  • the application discloses a method for preparing an emulsion as characterized in the appended claims comprising high energy methods.
  • the high energy method is microfluidization.
  • the high energy method is sonication.
  • the application discloses a method for labeling a cell, the method comprising contacting the cell ex vivo with an emulsion as characterized in the appended claims under conditions such that the fluorocarbon imaging reagent becomes associated with the cell.
  • the application discloses a method for detecting a cell in a subject, the method comprising: a) administering to the subject a cell that is labeled with an emulsion as characterized in the appended claims; and b) examining at least a portion of the subject by a nuclear magnetic resonance technique, thereby detecting the labeled cell in the subject.
  • a method for detecting transplanted cells in a transplant recipient comprising: a) administering cells for transplant to a transplant recipient, at least a portion of which cells for transplant are labeled with an emulsion of the application; b) examining at least a portion of the subject by a nuclear magnetic resonance technique, thereby detecting the labeled cells.
  • a method for quantifying cell number in vivo comprising: a) administering to the subject cells that are labeled with an emulsion of the application; b) examining at least a portion of the subject by a nuclear magnetic resonance technique, thereby detecting labeled cells in the subject; and c) quantifying the number of labeled cells in a region of interest (ROI).
  • ROI region of interest
  • a method for quantifying leukocyte number in vivo comprising: a) administering to the subject an emulsion of the application; b) extravesating a sample of peripheral blood from the subject and measure the effective cell loading of leukocytes; c) examining at least a portion of the subject by a nuclear magnetic resonance technique, thereby detecting labeled cells in the subject; and d) quantifying the number of labeled cells in a region of interest (ROI).
  • ROI region of interest
  • a method for labeling a cell comprising contacting the cell in vivo with an emulsion of the application under conditions such that the fluorocarbon imaging reagent becomes associated with the cell is also described herein.
  • a method for detecting a cell in a subject comprising: a) administering to the subject an emulsion of the application; and b) examining at least a portion of the subject by a nuclear magnetic resonance technique, thereby detecting the labeled cell in the subject.
  • the application discloses a method for measuring the partial pressure of oxygen in a tissue, the method comprising contacting the tissue in vivo with an emulsion as characterized in the appended claims under conditions such that the fluorocarbon imaging reagent becomes associated with the tissue.
  • the application discloses an emulsion as characterized in the appended claims for use in a method for detecting elevated vascular permeability in a tissue, wherein the tissue is to be contacted in vivo with the emulsion under conditions such that the fluorocarbon imaging reagent becomes associated with the tissue.
  • the application discloses a labeled cellular formulation for administration to a subject, the formulation comprising: a) a cell; and b) an emulsion as characterized in the appended claims that is associated with the cell.
  • the disclosure provides novel methods and reagents as characterized in the appended claims for labeling cells ex vivo with a nuclear magnetic resonance imaging reagent, such as a fluorocarbon imaging reagent, and quantifying the labeled cells in vivo or ex vivo.
  • Labeled cells may be detected by a 19 F nuclear magnetic resonance technique (e.g., MRI/MRS) and quantified according to methods described herein.
  • 19 F nuclear magnetic resonance techniques are excellent imaging tools for biological systems because of the absence of endogenous background signals. Fluorine is present, if at all, at exceedingly low levels in living organisms, and generally not in a chemical form that is detectable by liquid-state nuclear magnetic resonance techniques.
  • a method of the invention may comprise labeling cells ex vivo with a 19 F imaging reagent, administering the labeled cells to a subject, and detecting labeled cells in the subject.
  • the cells to be labeled may be a crude cellular fraction or tissue sample, or the cells may be cultured and/or subjected to enrichment prior to labeling.
  • particular cell types may be selected by fluorescence activated cell sorting (FACS) prior to labeling.
  • FACS fluorescence activated cell sorting
  • Other sorting or selective enrichment methods are known in the art for the various different cell types that may be of interest.
  • the types of cells that are labeled may also be controlled by the nature of the imaging reagent.
  • an imaging reagent may be formulated with or covalently bound to a targeting moiety that facilitates selective targeting of the imaging reagent to a particular population of cells. Imaging reagents are described further below. After labeling, cells may be immediately administered or the cells may be stored, further cultured, purified, enriched, segregated or processed in any way that is not incompatible with the intended use of such cells.
  • labeled cells may be administered for a therapeutic purpose.
  • Technology described herein may be used for monitoring the trafficking of cellular therapeutics in vivo or in any other desired milieu, such as a tissue explant.
  • Bone marrow cell transplants have been widely used for many years in recipients of ablative therapies for cancers.
  • Various purified cell populations have also been used in place of bone marrow, such as cell populations enriched for hematopoietic stem cells; for example cells may be harvested from umbilical cord blood or peripheral blood. After entering the bloodstream, the stem cells generally travel to the bone marrow, where they begin to produce new white blood cells, red blood cells, and platelets. This engraftment usually occurs within about 2 to 4 weeks after transplantation.
  • Non-myeloablative allogeneic transplantation i.e. reduced-intensity transplant
  • is a similar cell therapy that can be effective for treating several types of cancer.
  • this technique relies on a lower dose of radiation and/or chemotherapeutic and a limited graft-versus-host disease (the action of immune cells from the transplant against any residual host cancer cells) to provide sufficient anti-cancer activity, as well as the hematopoietic potential of the graft cells to restore the patient's hematopoietic system.
  • the techniques described herein may be used to monitor the locations and movements of graft cells in a non-myeloablative allogeneic transplantation.
  • Cellular therapeutics are also in development for use in the delivery of therapeutic proteins. It is further described herein that cells can be isolated, grown in quantity ex vivo and then implanted to produce and secrete soluble factors, which may be active either locally (e.g. enzymes, cytokines, and neurotransmitters) or at a distance (e.g. hormones and growth regulators). Cells may also be administered to a patient in order to accomplish complex therapeutic purposes, such as reconstitution of tissues, organs, or immune responses based on their ability to home to specific sites within the body, exit from the circulation, and integrate into surrounding tissue or differentiate to replace damaged tissue.
  • complex therapeutic purposes such as reconstitution of tissues, organs, or immune responses based on their ability to home to specific sites within the body, exit from the circulation, and integrate into surrounding tissue or differentiate to replace damaged tissue.
  • Stem cell therapies have also been proposed for myriad diseases including neurological disorders, particularly those characterized by cell death (e.g., Parkinson's disease, stroke and brain injury caused by trauma), cardiovascular disorders (e.g., myocardial infarction), muscle regeneration (e.g., in patients suffering from cachexia or other wasting disorders), pancreatic regeneration in diabetes, liver regeneration, etc.
  • cells, or a sub-population thereof may be labeled with an imaging reagent ex vivo prior to administration, thus allowing the monitoring of these cells in vivo.
  • In vivo monitoring by a nuclear magnetic resonance technique may be useful, for example, to evaluate the viability of the administered cells.
  • a doctor may tailor a dosing schedule depending on the degree to which labeled cells are detected in a patient after administration.
  • In vivo monitoring may also be useful in determining whether therapeutic cells have localized to a desired location.
  • the in vivo imaging of therapeutic cells may be used as a prognostic indicator that may be helpful in selecting the appropriate dosage, administration modes and additional therapeutic interventions that will benefit the patient.
  • Certain imaging advances described herein will benefit a broad range of cellular therapeutic strategies because these imaging methodologies will be able to detect when, where and if the therapeutic cells have been delivered to the desired targets in vivo. Additionally, the detection of labeled cells may be enhanced by quantification of labeled cells in a ROI, such as a particular organ or tissue.
  • DCs are known to be the most efficient antigen presenting cells and have the capacity to stimulate naive T cells to initiate an immune response. Because DCs are the most potent stimulators of immune response in the body, DCs represent a possible therapeutic approach to increasing the "visibility" of tumors to a patient's immune system. DCs are the focus of tumor vaccines in development. Varying methods are used to expose the dendritic cells to tumor antigens ex vivo, after which educated dendritic cells are reinfused to stimulate development of T-cell mediated tumor killing. Data applying a method described herein to the labeling and tracking of DCs and other cell types, are presented in WO2005072780 .
  • NK cells Natural killer cells
  • LAK cells lymphokine-activated killer cells
  • T cells which are white blood cells that attack pathogenic cells, has demonstrated promise against a variety of cancers, including pancreatic cancer, in which clinical trials are beginning, and against multiple sclerosis and HIV infection.
  • labeled cells may be administered to a subject for non-therapeutic purposes.
  • cells may be labeled ex vivo, administered to a subject and then detected, with the expectation that the labeled cells will behave similarly to like, unlabeled cells in vivo and may therefore be used to monitor the behavior of endogenous cell populations.
  • Monitoring may be used for the purpose of tracking movements of cells, particularly in the case of cells that are known to be highly mobile, such as cells of the immune system, many types of stem cells and blood born cells. Monitoring may also be used for the purpose of tracking viability or adherence of non-mobile cells at the site of implant.
  • Cells of many tissues such as muscle, liver, pancreas, kidney, brain or skin will tend to be relatively stationary, but disappearance of label may indicate a high death rate, low adherence, or other information.
  • Modern cell culture and sorting techniques allow the selective pooling and labeling of virtually any desired cell population, including various stem cell types, immune cell types, and other blood cell types.
  • cell surface markers can be used to sort mixed populations of cells to purify a population of interest.
  • both T cells and dendritic cells may be labeled ex vivo and detected in vivo.
  • labeled immune cells may be used as detectable proxies for the movements of immune cells in a patient.
  • Immune cells participate in and are markers for a host of inflammatory and autoimmune disorders, as well as cancer and atherosclerotic plaque formation.
  • any process involving the recruitment of immune cells may be detected in a patient by administering to the patient labeled immune cells.
  • the accumulation of label in a particular area provides an indication of the degree of immune response occurring in that portion of the body.
  • these types of studies involve histological techniques that are incompatible with living subjects. Certain methods of the disclosure may facilitate the development of therapeutic strategies for the treatment of human diseases.
  • the ability to track selected populations of immune cells non-invasively, and without the use of radioisotopes, can impact many areas of basic and clinical immunology, such as multiple sclerosis, diabetes, monitoring organ transplant rejection, and cancer. For instance, tumors are often highly infiltrated by immune cells. Labeled cells may be imaged in a subject to reveal the location of a tumor, and in some instances may be useful as a non-invasive detection screen. Early detection of cancers has been a critical problem, as most early stage cancers are readily treated by surgery without resort to debilitating chemotherapeutic agents. Likewise, the progress of other inflammatory diseases may be monitored by tracking the dynamics of immune cells in the patient. The effectiveness of immunosuppressant therapy may be assessed as well.
  • the recipient could receive a dose of labeled immune cells prior to receiving the transplantation. In vivo monitoring of the accumulation of immune cells in the transplant could then be used as an early warning sign of rejection.
  • the methods disclosed herein are particularly desirable because the alternative, biopsies, are well-known to increase the risk of organ rejection.
  • cells for use in a bone marrow cell transplant, or a peripheral blood stem cell transplant may be labeled ex vivo as described herein, administered, and monitored in vivo by a nuclear magnetic resonance technique. Such monitoring may be used to evaluate the engraftment of donor cells in the recipient bone cavities, as well as survivorship and movement of labeled cells in the recipient.
  • a physician can use information relating to the trafficking of donor cells in a recipient as an early indication of the likely success or failure of the procedure. This type of early detection will allow physicians to tailor the post-transplant therapeutic regimen accordingly.
  • Another cellular cancer therapeutic where the detection technology can be applied is the allogeneic non-myeloablative, or reduced intensity transplant.
  • This procedure may be used with a donor lymphocyte infusion to boost graft-versus-tumor effect which destroys cancer cells.
  • the entire population, or a fraction, of transplanted cells could be labeled before infusion.
  • a nuclear magnetic resonance technique could then be used determine where the cells traffic to in the body, which can be indicative of the efficacy of the procedure.
  • the cell's trafficking pattern may be used to calibrate dose.
  • cells involved in formation of new tissue can be labeled, administered to a subject, and detected to identify hotspots of tissue formation.
  • smooth muscle cells and/or endothelial precursor cells may be labeled and introduced into the bloodstream.
  • Such cells are expected to accumulate at sites of angiogenic activity.
  • Angiogenic activity may be associated with physiological and pathological events such as menstrual cycling, early pregnancy, collateral vessel formation in response to arterial blockages, tumor development and wound healing.
  • cells involved in wound healing such as fibroblasts, may be labeled and administered systemically or to a site of suspected injury in order to monitor cellular behavior.
  • a medicament or delivery device containing labeled cardiomyocyte lineage cell aggregates or cells derived therefrom may be provided for treatment of a human or animal body, including formulations for cardiac therapy.
  • Cardiomyocyte lineage cells may be administered to a patient in a method for reconstituting or supplementing contractile and/or pacemaking activity in cardiac tissue (see US Patent Application No. 20060040389 , 20050112104 , 20050244384 ,).
  • labeled cardiomyocyte lineage cells may be used to regenerate or repair striated cardiac muscle that has been damaged through disease or degeneration.
  • the labeled cardiomyocyte lineage cells integrate with the healthy tissue of the recipient to replace the function of the dead or damaged cells, thereby regenerating the cardiac muscle as a whole. Cardiac muscle does not normally have reparative potential.
  • the labeled cardiomyocyte lineage cells are used, for example, in cardiac muscle regeneration for a number of principal indications: (i) ischemic heart implantations, (ii) therapy for congestive heart failure patients, (iii) prevention of further disease for patients undergoing coronary artery bypass graft, (iv) conductive tissue regeneration, (v) vessel smooth muscle regeneration and (vi) valve regeneration.
  • the administration of the cells can be directed to the heart, by a variety of procedures such as localized administration.
  • the mesenchymal stem cells can be from a spectrum of sources including, in order of preference: autologous, allogeneic, or xenogeneic. Monitoring of the progress of these cells in vivo is also described herein.
  • the cardiomyocyte lineage cells may be cardiomyocyte precursor cells, or differentiated cardiomyocytes.
  • Differentiated cardiomyocytes include one or more of primary cardiomyocytes, nodal (pacemaker) cardiomyocytes; conduction cardiomyocytes; and working (contractile) cardiomyocytes, which may be of atrial or ventricular type.
  • cells may come from a muscle sample (or other sample) that contains muscle progenitor cells such as satellite cells (see US Patent Application No. 20050244384 ). It is also described herein that cells may be mesenchymal stem cells (MSCs) (see US Patent Application No. 20050112104 ).
  • MSCs mesenchymal stem cells
  • a “cardiomyocyte precursor” is defined as a cell that is capable (without dedifferentiation or reprogramming) of giving rise to progeny that include cardiomyocytes. Such precursors may express markers typical of the lineage, including, without limitation, cardiac troponin I (cTnI), cardiac troponin T (cTnT), sarcomeric myosin heavy chain (MHC), GATA4, Nkx2.5, N-cadherin, .beta.1-adrenoceptor (.beta.1-AR), ANF, the MEF-2 family of transcription factors, creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (ANF).
  • cTnI cardiac troponin I
  • cTnT cardiac troponin T
  • MHC sarcomeric myosin heavy chain
  • GATA4 sarcomeric myosin heavy chain
  • GATA4 sarcomeric myosin heavy chain
  • cells may prove to be so thoroughly associated with a biological site or structure of interest that the labeled cells may be administered for the sole purpose of aiding in the visualization of such a structure.
  • immune cells characteristically infiltrate tumors. Accordingly, labeled immune cells may be administered for the purpose of visualizing tumors.
  • Various animal models of diseases may evince altered dynamics or survival of one or more cell populations. Such cell populations may be labeled, administered to the animal and monitored. For example, the infiltration of immune cells into the pancreas of the NOD mouse model for diabetes may be monitored.
  • Other examples of animal models include: experimental allergic encephalomyelitis (multiple sclerosis model), gliosarcoma tumor models, and organ-transplant rejection.
  • a drug screening assay may comprise administering labeled cells to an animal and detecting the cells in vivo in the presence of a test agent. Changes in cell behavior that are correlated with the presence of the test agent may be indicative of a therapeutic effect. Such changes may be detected by comparison to a suitable reference, including, for example, the same animal before and after treatment with the test agent or a separate, untreated animal.
  • the methods may be used to evaluate the effects of test conditions, such as an exercise regimen, injury, genetic alteration, etc. As an example, it is expected that a drug for treatment of an autoimmune disease would decrease the tendency of immune cells to accumulate in an affected tissue.
  • methods disclosed herein may be used to evaluate kinetic properties of cells, such as the rate at which cells arrive at a particular site and the time of signal persistence at a site.
  • Drug screening assays may be particularly powerful when combined with in vivo monitoring of tightly defined cell populations, such as certain groups of immune cells that are implicated in various disorders. For example, monitoring of labeled cytotoxic T cells may be particularly useful in identifying drugs that may be useful in preventing transplant rejection.
  • the ability to monitor cells in vivo provides a powerful assay that may be applied to the analysis of essentially any experimental animal, including, for example, any of the various transgenic or otherwise mutant mice that have been generated.
  • SPIO agents for example, impart contrast to conventional 1 H images by locally perturbing the magnetic field experienced by the nearby mobile water molecules, which in turn modulates T 1 , T 2 , or T 2 *.
  • Methods described herein are distinctly different from all methods using metal-ion based contrast agents because signals from 19 F nuclei in the imaging reagents may be directly detected and, optionally, imaged.
  • compositions of the application may find application in 19 F-MR spectroscopy (MRS), imaging (MRI), and spectroscopic imaging (MRSI).
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • MRSI spectroscopic imaging
  • the emulsions of the application may be used for in situ labeling of resident macrophages and monocytes.
  • a bolus of emulsion is directly injected intravenously (iv).
  • the emulsion is buffered appropriately (e.g., a physiologically safe pH, osmality, etc.) for safe iv injection.
  • the emulsion droplets are scavenged from the blood by resident phagocytic cells, such as neutrophils, macrophages and monocytes. These labeled cells participate in inflammatory events within the body.
  • An emulsion bolus may be injected iv, and the emulsion droplets are taken up by resident phagocytes that migrate to a tumor.
  • the detection of 19 F at the tumor is a diagnostic tool which identifies the location of the tumor as well as the distribution and extent of its macrophage or inflammation activity.
  • perfluoro-15-crown-5 ether a principal component of the emulsions, is known to coordinate or bind oxygen. It is known in the art that this molecule, in the presence of oxygen, has its 19 F NMR relaxation times (T1, T2 and T2*) shortened by an amount that is linearly proportional to the local partial pressure of oxygen (pO2).
  • T1 measurements of the interstitial and macrophage-incorporated perfluoro-15-crown-5 ether in the tumor may be used to measure pO2, which can be a sensitive marker of the efficacy of a variety of cancer therapeutics.
  • Emulsions of the application may be used to assay hypoxia or hyperoxia.
  • pO2 sensing of tissues may be achieved by direct injection of emulsions into tissue.
  • the emulsions of the application may be used to detect a wide range of lesions and diseases where inflammation is presentFurther, it is described herein that the disease may be selected from the group consisting of cancer, cardiovascular disease, inflammatory bowel disease, autoimmune disease (e.g., multiple sclerosis, rheumatoid arthritis, type-1 diabetes, lupus, Crohn's disease, optic neuritis, etc.), organ transplant rejection, infectious diseases, and traumatic brain and spinal cord injury.
  • autoimmune disease e.g., multiple sclerosis, rheumatoid arthritis, type-1 diabetes, lupus, Crohn's disease, optic neuritis, etc.
  • organ transplant rejection e.g., multiple sclerosis, rheumatoid arthritis, type-1 diabetes, lupus, Crohn's disease, optic neuritis, etc.
  • Emulsions of the application may be used to image lesions of the liver via a 19 F image of the emulsion distribution in that organ.
  • Emulsions of the application which are injected iv may be cleared by the liver and taken up by liver cells. Lesions of the liver may result in anomalous and heterogeneous distibutions of emulsion uptake and 19 F image intensity.
  • the emulsions of the application may be used as a probe of tissue oxygenation.
  • the emulsions of the application can serve as a sensor for pO2 in tissue using 19 F MRS/MRI.
  • Perfluoro-15-crown-5 ether coordinates oxygen, thereby changing its 19 F T1-value several fold.
  • a measurement of the 19 F T1 either by MRS or MRI e.g., using an image map of T1I
  • Emulsions of the application may accumulate in areas with elevated vascular permeability, such as, e.g., in tumors.
  • Emulsions of the application may be used to assay the perfusion of tissues, provide the possibility of determining the blood volumes in tissues, to selectively shorten the relaxation times or densities of the blood and to graphically visualize the permeability of blood vessels.
  • Emulsions of the application may be used for specific diagnosis of malignant tumors, early therapy control in cytostatic, antiphlogistic or vasodilatative therapy, early detection of underperfused areas (e.g., in the myocardium), angiography in vascular diseases, and detection and diagnosis of sterile or infectious inflammations.
  • the emulsions of the application may be used as an artificial oxygen carrier or artificial blood substitute (See US Patent Publication No. 20040057906 , US Patent Nos. 4838,274 and 5,785,950 and WO 96/40057 ).
  • the emulsions may be used in vivo or ex vivo.
  • the emulsions of the disclosure are capable of having dissolved in them large amounts of gases, including oxygen, carbon dioxide, and air, per unit volume. Accordingly, fluorocarbons (FCs) and perfluorocarbons (PFCs) may be used as carriers in applications wherein oxygen must be supplied to organs and tissues.
  • the imaging reagent used in the subject methods is a fluorocarbon, i.e., a molecule including at least one carbon-fluorine bond.
  • the imaging reagents disclosed herein may be detected by 19 F MRI and other nuclear magnetic resonance techniques, such as MRS techniques.
  • a fluorocarbon imaging reagent may have one or more of the following properties: 1) reduced cytotoxicity; 2) a 19 F NMR spectrum that is simple, ideally having a single, narrow resonance to minimize chemical shift artifacts; 3) high sensitivity with a large number of NMR-equivalent fluorine atoms in each molecule; 4) formulated to permit efficient labeling of many cell types and not restricted to phagocytic cells.
  • the imaging reagent comprises a plurality of fluorines bound to carbon, e.g., greater than 5, greater than 10, greater than 15 or greater than 20 fluorines bound to carbon.
  • at least 4, at least 8, at least 12 or at least 16 of the fluorines have a roughly equivalent NMR chemical shift.
  • the imaging reagents can be employed in one or more of at least three modalities: 1) imaging reagents that are internalized or otherwise absorbed by target cells without the formation of any covalent or other binding association; 2) imaging reagents that covalently attach to target cells; and 3) imaging reagents coupled to molecules, such as antibodies or ligands, that bind to molecules present on the target cells.
  • Imaging reagents of the first type include the perfluoro crown ethers and other perfluoropolyethers (PFPEs) that are taken up by cells and, preferably, are retained in the cell without degradation for a substantial period of time, e.g., having a half-life in the cell of at least 1 hour, at least 4 hours, at least about a day, at least about three days, or even at least about a week.
  • PFPEs perfluoro crown ethers and other perfluoropolyethers
  • Imaging reagents of the second type include electrophilic compounds that react with nucleophilic sites on the cell surface, such as exposed thiol, amino, and/or hydroxyl groups. Accordingly, imaging reagents such as maleimides, alkyl iodides, N-hydroxysuccinimide or N-hydroxysulfosuccinimide esters (NHS or sulfo-NHS esters), acyl succinimides, and the like can form covalent bonds with cell surfaces.
  • Other techniques used in protein coupling can be adapted for coupling imaging reagents to cell surface proteins. See Means et al. (1990) Bioconjugate Chemistry 1:2-12 , for additional approaches to such coupling.
  • Imaging reagents of the third type can be prepared by reacting imaging reagents of the second type not with the cells themselves, but with a functional moiety that is a cell-targeting ligand or antibody.
  • Suitable ligands and antibodies can be selected for the application of interest.
  • a ligand that selectively targets hematopoietic cells could be labeled with an imaging reagent as described herein and administered to a patient, such as by injection.
  • an imaging reagent can be coupled to an indiscriminate internalizing peptide, such as antepennepedia protein, HIV transactivating (TAT) protein, mastoparan, melittin, bombolittin, delta hemolysin, pardaxin, Pseudomonas exotoxin A, clathrin, Diphtheria toxin, C9 complement protein, or a fragment of any of these.
  • an imaging reagent can be coupled to an indiscriminate internalizing peptide, such as antepennepedia protein, HIV transactivating (TAT) protein, mastoparan, melittin, bombolittin, delta hemolysin, pardaxin, Pseudomonas exotoxin A, clathrin, Diphtheria toxin, C9 complement protein, or a fragment of any of these.
  • TAT HIV transactivating
  • the internalizing peptide may be derived from the drosophila antepennepedia protein, or homologs thereof.
  • the 60-amino acid-long homeodomain of the homeoprotein antepennepedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is coupled. See for example Derossi et al. (1994) J Biol Chem 269:10444-10450 ; and Perez et al. (1992) J Cell Sci 102:717-722 . It has been demonstrated that fragments as small as 16 amino acids long of this protein are sufficient to drive internalization. See Derossi et al. (1996) J Biol Chem 271:18188-18193 .
  • TAT HIV transactivator
  • This protein appears to be divided into four domains ( Kuppuswamy et al. (1989) Nucl. Acids Res. 17:3551-3561 ).
  • Purified TAT protein is taken up by cells in tissue culture ( Frankel and Pabo, (1989) Cell 55:1189-1193 ), and peptides, such as the fragment corresponding to residues 37 -62 of TAT, are rapidly taken up by cell in vitro ( Green and Loewenstein, (1989) Cell 55:1179-1188 ).
  • the highly basic region mediates internalization and targeting of the internalizing moiety to the nucleus ( Ruben et al., (1989) J. Virol. 63:1-8 ).
  • Peptides or analogs that include a sequence present in the highly basic region can be conjugated to fluorinated imaging reagents to aid in internalization and targeting those reagents to the intracellular milieu.
  • the present invention provides novel compositions as characterized in the appended claims comprising imaging reagents.
  • the present invention provides an aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, an emulsifier, a surfactant co-mixture, and an additive, wherein the emulsifier comprises glycerol polyethylene glycol ricinoleate.
  • the surfactant co-mixture comprises lecithin (i.e., lipoid egg phosphatidyl choline), cholesterol, and dipalmitoyl phosphatidylethanolamine (DPPE).
  • the surfactant co-mixture comprises 70 mol % of lecithin; 28 mol % of cholesterol; and 2 mol % of DPPI.
  • the additive is propylene glycol.
  • the emulsifier also described herein may be a non-ionic solubiliser.
  • the emulsifier comprises glycerol polyethylene glycol ricinoleate.
  • the emulsifier further comprises fatty acid esters of polyethylene glycol, free polyethylene glycols, and ethoxylated glycerol.
  • the emulsifier is prepared by reacting castor oil and ethylene oxide in a molar ratio of 1:35.
  • Exemplary emulsifiers can be obtained from BASF Corporation and are sold under the trade name of Cremophor® EL.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) comprises perfluor-15-crown-5 ether or PFPE oxide in the range of 20% to 50% w/v, such as 25% to 45% w/v, such as 30% to 40% w/v, such as 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture(e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) comprises perfluor-15-crown-5 ether or PFPE oxide in the range of 35% to 36% w/v, such as 35.1%, 35.2%, 35.3%, 35.4%, 35.5%, 35.6%, 35.7%, 35.8%, or 35.9% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) comprises perfluor-15-crown-5 ether or PFPE oxide in 35.6% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) comprises Cremophor® EL in the range of 1% to 10% w/v, such as 1% to 5% w/v, such as 1%, 2%, 3%, 4%, or 5% w/v.
  • Cremophor® EL in the range of 1% to 10% w/v, such as 1% to 5% w/v, such as 1%, 2%, 3%, 4%, or 5% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) comprises Cremophor® EL in 3% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide), Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and propylene glycol comprises propylene glycol in 2% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, an additive (e.g., propylene glycol), and a surfactant co-mixture, wherein the surfactant co-mixture comprises lecithin, cholesterol, and DPPE, comprises the surfactant co-mixture, wherein the surfactant co-mixture comprises lecithin, cholesterol, and DPPE, in the range of 1% to 10% w/v, such as 1% to 5% w/v, such as 1%, 2%, 3%, 4%, or 5% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, an additive (e.g., propylene glycol), and a surfactant co-mixture, wherein the surfactant co-mixture comprises lecithin, cholesterol, and DPPE, comprises the surfactant co-mixture, wherein the surfactant co-mixture comprises lecithin, cholesterol, and DPPE, in 2% w/v.
  • an additive e.g., propylene glycol
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) further comprises polyethylamine.
  • the aqueous composition comprises polyethylamine in the range of 0.01% to 5.0% w/w.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), an additive (e.g., propylene glycol), and polyethylamine further comprises protamine sulfate.
  • the aqueous composition further comprises protamine sulfate in the range of 0.01 % to 5.0% w/w.
  • the present invention provides an aqueous composition
  • aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide in 35.6% w/v, Cremophor® EL in 3.0% w/v, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE) in 2.0% w/v, and an additive (e.g., propylene glycol) in 2.0% w/v.
  • a surfactant co-mixture e.g., comprising lecithin, cholesterol, and DPPE
  • an additive e.g., propylene glycol
  • the terms emulsion and nanoemulsion as used in this application are equivalent unless specifically stated otherwise.
  • the emulsion may further comprise a block copolymer of polyethylene and polypropylene glycol.
  • the emulsion may further comprise a PluronicTM.
  • Nonionic PluronicTM surfactants polyethyleneoxide (PEO)/polypropyleneoxide (PPO)/polyethyleneoxide (PEO) block (ABA type), (PEO/PPO/PEO) block copolymers, exhibit a wide range of hydrophilicity/ hydrophobicity as a function of the PEO/PPO ratio, so that one can expect to obtain different phase separated morphologies with polymers such as PLA as well as different degrees of hydration of the matrix. In particular, hydration plays an important role in determining polymer degradation via hydrolysis of the ester backbone.
  • emulsions of the present invention further comprise tri-block copolymer which comprises polyethyleneoxide and polypropyleneoxide.
  • emulsions of the present invention comprise a tri-block copolymer of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) comprising 80% PEO content.
  • Exemplary tri-block copolymers can be obtained from BASF Corporation and are sold under the trade name of PluronicTM F68.
  • aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68 is also described herein.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68 may comprise perfluoro-15-crown-5 or PFPE oxide ether in the range of 10% to 20% w/w, such as 12% to 17% w/w, such as 12%, 13%, 14%, 15%, 16%, or 17% w/w.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68 may comprise perfluoro-15-crown-5 ether or PFPE oxide in 15% w/w and that the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68, may comprise the PluronicTM F68 in the range of 0.1% to 2.0% w/w, such as 0.1 % to 1.0% w/w, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% w/w. Moreover, it is described herein that the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68, may comprise the PluronicTM F68 in 0.6% w/w.
  • aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide in 15% w/w and the PluronicTM F68 in 0.6% w/w is also described herein.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68 may further comprise protamine sulfate. It is also described herein that the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, the PluronicTM F68, and protamine sulfate may comprise protamine sulfate in the range of 0.01% to 1.0% w/w, such as 0.01% to 0.5% w/w, such as 0.01% to 0.10% w/w, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10% w/w.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, the PluronicTM F68, and protamine sulfate may comprise protamine sulfate in 0.04% w/w.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68 further may comprise polyethylamine.
  • An aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide in 15% w/w, the PluronicTM F68 in 0.6% w/w, and protamine sulfate in 0.04% w/w is also described herein.
  • the present invention also provides formulations of the compositions of the present invention as characterized in the appended claimsthat are suitable for uptake by cells.
  • the compositions of the present invention may be formulated as an emulsion.
  • the present invention provides an emulsion comprising an aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture, and an additive.
  • the surfactant co-mixture comprises lecithin, cholesterol, and dipalmitoyl phosphatidyl ethanolamine (DPPE).
  • DPPE dipalmitoyl phosphatidyl ethanolamine
  • the surfactant co-mixture comprises 70 mol % of lecithin; 28 mol % of cholesterol; and 2 mol % of DPPI.
  • the additive is propylene glycol.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) comprises perfluor-15-crown-5 ether or PFPE oxide in the range of 20% to 50% w/v, such as 25% to 45% w/v, such as 30% to 40% w/v, such as 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) comprises perfluor-15-crown-5 ether or PFPE oxide in the range of 35% to 36% w/v, such as 35.1%, 35.2%, 35.3%, 35.4%, 35.5%, 35.6%, 35.7%, 35.8%, or 35.9% w/v.
  • a surfactant co-mixture e.g., comprising lecithin, cholesterol, and DPPE
  • an additive e.g., propylene glycol
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) comprises perfluor-15-crown-5 ether or PFPE oxide in 35.6% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) comprises Cremophor® EL in the range of 1% to 10% w/v, such as 1% to 5% w/v, such as 1%, 2%, 3%, 4%, or 5% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and an additive (e.g., propylene glycol) comprises Cremophor® EL in 3% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and propylene glycol comprises propylene glycol in the range of 1% to 10% w/v, such as 1% to 5% w/v, such as 1%, 2%, 3%, 4%, or 5% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE), and propylene glycol comprises propylene glycol in 2% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, an additive (e.g., propylene glycol), and a surfactant co-mixture
  • the surfactant co-mixture comprises lecithin, cholesterol, and DPPE
  • the surfactant co-mixture comprises lecithin, cholesterol, and DPPE, in the range of 1% to 10% w/v, such as 1% to 5% w/v, such as 1%, 2%, 3%, 4%, or 5% w/v.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, Cremophor® EL, an additive (e.g., propylene glycol), and a surfactant co-mixture, wherein the surfactant co-mixture comprises lecithin, cholesterol, and DPPE, comprises the surfactant co-mixture, wherein the surfactant co-mixture comprises lecithin, cholesterol, and DPPE, in 2% w/v.
  • an additive e.g., propylene glycol
  • the present invention provides an emulsion comprising an aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide in 35.6% w/v, Cremophor® EL in 3.0% w/v, a surfactant co-mixture (e.g., comprising lecithin, cholesterol, and DPPE) in 2.0% w/v, and an additive (e.g., propylene glycol) in 2.0% w/v.
  • a surfactant co-mixture e.g., comprising lecithin, cholesterol, and DPPE
  • an additive e.g., propylene glycol
  • an emulsion comprising an aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68 is also described herein. Further, it is described herein that in the foregoing emulsion, the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68, may comprise perfluoro-15-crown-5 ether or PFPE oxide in the range of 10% to 20% w/w, such as 12% to 17% w/w, such as 12%, 13%, 14%, 15%, 16%, or 17% w/w.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68 may comprise perfluoro-15-crown-5 ether or PFPE oxide in 15% w/w.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68 may comprise the PluronicTM F68 in the range of 0.1% to 2.0% w/w, such as 0.1% to 1.0% w/w, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% w/w. It is further described herein that in the foregoing emulsion the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68 may comprises the PluronicTM F68 in 0.6% w/w.
  • An emulsion comprising an aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide in 15% w/w and the PluronicTM F68 in 0.6% w/w is also described herein.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide and the PluronicTM F68 may further comprise protamine sulfate.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, the PluronicTM F68 and protamine sulfate may comprise protamine sulfate in the range of 0.01% to 1.0% w/w, such as 0.01% to 0.5% w/w, such as 0.01% to 0.10% w/w, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10% w/w.
  • the aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide, the PluronicTM F68, and protamine sulfate may comprise protamine sulfate in 0.04% w/w.
  • An emulsion comprising an aqueous composition comprising perfluoro-15-crown-5 ether or PFPE oxide in 15% w/w, the PluronicTM F68 in 0.6% w/w, and protamine sulfate in 0.04% w/w is also described herein.
  • compositions and emulsions of the present invention comprise Cremophor® EL, a nonionic solubiliser and emulsifier comprising polyethylene glycol ricinoleate, made by reacting castor oil with ethylene oxide in a molar ratio of 1:35.
  • Cremophor® EL a nonionic solubiliser and emulsifier comprising polyethylene glycol ricinoleate, made by reacting castor oil with ethylene oxide in a molar ratio of 1:35.
  • This material can be obtained from BASF Corporation.
  • the emulsion may further comprise a lipid.
  • the lipid is DMPC.
  • the emulsion further comprises a PluronicTM.
  • the PluronicTM is F68.
  • the emulsion may further comprise polyethylamine.
  • the emulsion may further comprise protamine sulfate.
  • the emulsion further comprises a PluronicTM.
  • the PluronicTM is F68.
  • the emulsion of the present invention further comprises protamine sulfate.
  • Emulsions of the present invention will preferably have a distribution of droplet sizes that allow adequate cellular uptake.
  • a uniform droplet size may be advantageous.
  • the desired degree of uniformity of droplet size may vary depending upon the application.
  • the emulsion has a mean droplet size less than 500 nm, or less than 400 nm, or less than 300 nm, or less than 200 nm in diameter.
  • 25%, or 50%, or 75% or more of the droplets will fall within the selected range.
  • Droplet sizes may be evaluated by, for example, light scattering techniques or by visualizing the emulsion droplets using EM micrographs.
  • the emulsions have a mean droplet size of less than 200 nm, or less than 100 nm, or less than 50 nm in diameter.
  • small droplet size is advantageous. In certain embodiments, small droplet size increases circulation time in applications where the emulsion is injected iv. In certain embodiments, droplets are seperable from cells by circulation. In certain embodiments, small droplet size increases ex vivo cell labeling. In certain embodiments, small droplet size increases uniform labeling.
  • Emulsions for use in cells should preferably be stable at a wide range of temperatures.
  • emulsions will be stable at body temperature (37 °C for humans) and at a storage temperature, such as 4 °C or room temperature (20-25 °C).
  • a storage temperature such as 4 °C or room temperature (20-25 °C).
  • the emulsion will experience a temperature of about 37 °C.
  • a preferred emulsion will retain the desired range of droplet sizes at temperatures ranging from refrigeration temperatures up to body temperature.
  • the emulsion is stable at temperatures ranging from 4 °C to 37 °C.
  • the emulsion has a polydispersity index ranging from 0.1 to 0.2.
  • the properties of an emulsion may be controlled primarily by the properties of the imaging reagent itself, the nature of surfactants and/or solvents used, and the type of processing device (e.g., sonicator, Microfluidizer, homogenizer, etc.). Methods for forming emulsions with certain PFPE molecules are extensively described in U.S. Pat. Nos. 5,330,681 and 4,990,283 . A continuous phase of a polyhydroxylated compound, such as polyalcohols and saccharides in concentrated aqueous solution may be effective.
  • the following polyalcohols and saccharides have proved to be particularly effective: glycerol, xylitol, mannitol, sorbitol, glucose, fructose, saccharose, maltitol, dimer compounds of glycerol (di-glycerol or bis(2,3-di-hydroxypropyl) ether, solid water soluble polyhydroxylated compounds as sugars and glycerol condensation products as triglycerol and tetraglycerol.
  • the dispersion in emulsion may be performed in the presence of conventional surfactants, including cationic, anionic, amphoteric and non-ionic surfactants.
  • surfactants include sodium lauryl sulphate, sulphosuccinate (sulphosuccinic hemiester), coco-amphocarboxyglycinate, potassium cetyl phosphate, sodium alkyl-polyoxyethylene-ether carboxylate, potassium benzalconium chloride, alkyl amidopropyl betaine, cetyl-stearilic ethoxylated alcohol, and sorbitan-ethoxylate(20)-mono-oleate Tween 20. While thermodynamic equations may be used to attempt to predict mixtures of imaging reagents that will give emulsions having the desired droplet sizes and stability, it is generally accepted that actual testing of various mixtures will be most effective. The emulsification of mixtures is simple and quick, permitting rapid testing of a wide range of combinations to identify those that give rise to emulsions that are suitable for use in the methods disclosed herein.
  • preferred emulsions are designed to facilitate uptake of the imaging reagent by the subject cells.
  • a surfactant may be designed to form stable emulsions that carry a large quantity of perfluoro-15-crown-5 ether or PFPE oxide into the aqueous phase. Additionally, it may have properties that increase the intracellular delivery of the emulsion droplets in the shortest possible incubation time. Increasing the perfluoro-15-crown-5 ether or PFPE oxide intracellular loading improves sensitivity to the labeled cells. Furthermore, minimizing the culture time can be important when working with the primary cells cultures. The efficiency of intracellular uptake depends on cell type.
  • the uptake efficiency can be boosted substantially by designing the surfactant so that the surface of the emulsion droplet has properties that promote cellular uptake in culture (i.e. "self-delivering" emulsion droplets) (see Janjic et al, JACS, 2008, 130 (9), 2832 -2841 and US Provisional Patent Application 61/062,710 ).
  • the emulsion droplet surface can be made to have lipophilic, or optionally cationic, properties via appropriate surfactant design.
  • the surfactant can incorporate lipids, such as cationic or neutral lipids, oil-in-water emulsions, micelles, mixed micelles, or liposomes, that tend to bind to or fuse with the cell's surface, thereby enhancing emulsion droplet uptake.
  • lipids such as cationic or neutral lipids, oil-in-water emulsions, micelles, mixed micelles, or liposomes, that tend to bind to or fuse with the cell's surface, thereby enhancing emulsion droplet uptake.
  • the emulsion droplet surface may also incorporate cell delivery signals such as polyamines. Examples include emulsions that have polyamines, such as polyethylenimine or protamine sulfate, incorporated into the emulsion droplet's surfactant layer during processing.
  • a colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle).
  • a liposome i.e., an artificial membrane vesicle.
  • Suitable cationic lipids are described in the following: Felgner et al., 1987, PNAS 84, 7413-7417 ; Eppstein et al., U.S. Pat. No. 4,897,355 ), ( Rose, U.S. Pat. No. 5,279,833 ; Eppand et al. U.S. Pat. No. 5,283,185 ; Gebeyehu et al., U.S. Pat. No.
  • colloidal dispersion systems are used, such as macromolecule complexes, nanocapsules, microspheres, and beads.
  • emulsions have "self-delivering" properties without having to add uptake enhancing reagents.
  • Said emulsions are preferably stable and have a shelf-life of a period of months or years.
  • surfactants and uptake enhancing reagents are not meant to be exclusive groups and in some cases they may be overlapping.
  • Methods described herein may be used with a wide range of cells, including both prokaryotic and eukaryotic cells, and preferably mammalian cells.
  • Technologies for cell preparation include cell culture, cloning, nuclear transfer, genetic modification and encapsulation, wherein cloning does not encompass processes for cloning human beings.
  • a partial list of suitable mammalian cells includes: blood cells, myoblasts, bone marrow cells, peripheral blood cells, umbilical cord blood cells, cardiomyocytes (and precursors thereof), chondrocytes (cartilage cells), dendritic cells, fetal neural tissue, fibroblasts, hepatocytes (liver cells), islet cells of pancreas, keratinocytes (skin cells) and stem cells.
  • the cells to be used are a fractionated population of immune cells.
  • lymphocytes such as B lymphocytes (Fc receptors, MHC class II, CD19+, CD21+), hELer T lymphocytes (CD3+, CD4+, CD8-), cytolytic T lymphocytes (CD3+, CD4-, CD8+), natural killer cells (CD16+), the mononuclear phagocytes, including monocytes, neutrophils and macrophages, and dendritic cells.
  • lymphocytes such as B lymphocytes (Fc receptors, MHC class II, CD19+, CD21+), hELer T lymphocytes (CD3+, CD4+, CD8-), cytolytic T lymphocytes (CD3+, CD4-, CD8+), natural killer cells (CD16+), the mononuclear phagocytes, including monocytes, neutrophils and macrophages, and dendritic cells.
  • Other cell types that may be of interest include eosinophils and basophils.
  • Cells may be autologous (i.e., derived from the same individual) or syngeneic (i.e., derived from a genetically identical individual, such as a syngeneic littermate or an identical twin), although allogeneic cells (i.e., cells derived from a genetically different individual of the same species) are also contemplated.
  • allogeneic cells i.e., cells derived from a genetically different individual of the same species
  • xenogeneic (i.e., derived from a different species than the recipient) cells such as cells from transgenic pigs, may also be administered.
  • the donor cells are xenogeneic, it is preferred that the cells are obtained from an individual of a species within the same order, more preferably the same superfamily or family (e.g. when the recipient is a human, it is preferred that the cells are derived from a primate, more preferably a member of the superfamily Hominoidea).
  • Cells may, where medically and ethically appropriate, be obtained from any stage of development of the donor individual, including infant (e.g., from birth to approximately three years of age in humans), child (e.g.. from about three years of age to about 13 years of age in humans), adolescent (e.g., from about 13 years of age to about 18 years of age in humans), young adult (e.g., from about 18 years of age to about 35 years of age in humans), adult (from about 35 years of age to about 55 years of age in humans) or elderly (e.g., from about 55 years and beyond of age in humans), wherein the cells are not derived from a human embryo.
  • infant e.g., from birth to approximately three years of age in humans
  • child e.g.. from about three years of age to about 13 years of age in humans
  • adolescent e.g., from about 13 years of age to about 18 years of age in humans
  • young adult e.g., from about 18 years of age to about 35
  • cells are labeled by contacting the cells with an emulsion of the imaging reagent, such that the reagent is taken up by cells.
  • Both phagocytic and non-phagocytic cells may be labeled by such a method.
  • both dendritic cells (phagocytic) and gliosarcoma cells (non-phagocytic) can be labeled by contacting the cells with an emulsion of the imaging reagent.
  • a method of the invention may comprise labeling cells in vivo with a 19 F imaging reagent and detecting labeled cells in the subject.
  • the cells to be labeled may be determined by specific properties of the cells such as phagocytic activity.
  • the cells that are labeled may be controlled by the route of administration of the imaging reagent.
  • the types of cells that are labeled may be controlled by the nature of the imaging reagent. For example, simple colloidal suspensions of imaging reagent will tend to be taken up more quickly by cells with phagocytic activity.
  • an imaging reagent may be formulated with or covalently bound to a targeting moiety that facilitates selective targeting of the imaging reagent to a particular population of cells.
  • the imaging reagent comprises perfluoro-15-crown ether.
  • the cells to be labeled are stem cells, wherein the stem cells are not human embryonic stem cells.
  • Stem cell therapies are commonly used as part of an ablative regimen for treatment of cancer with high dose radiation and/or chemotherapeutic agents.
  • Ablative regimens generally employ hematopoietic stem cells, or populations of cells containing hematopoietic stem cells, as may be obtained, for example, from peripheral blood, umbilical cord blood or bone marrow. Cells of this type, or a portion thereof, may be labeled and tracked in vivo to monitor survival and engraftment at the appropriate location.
  • Other types of stem cells are increasingly attractive as therapeutic agents for a wide variety of disorders.
  • cells may be mouse embryonic stem cells, or ES cells from another model animal.
  • the labeling of such cells may be useful in tracking the fate of such cells administered to mice, optionally as part of a preclinical research program for developing embryonic stem cell therapeutics.
  • mouse embryonic stem cells include: the JM1 ES cell line described in M. Qiu et al., Genes Dev 9, 2523 (1995 ), and the ROSA line described in G. Friedrich, P. Soriano, Genes Dev 5, 1513 (1991 ), and mouse ES cells described in US Patent No. 6,190,910 . Many other mouse ES lines are available from Jackson Laboratories (Bar Harbor, Maine).
  • human embryonic stem cells examples include those available through the following suppliers: Arcos Bioscience, Inc., Foster City, California, CyThera, Inc., San Diego, California, BresaGen, Inc., Athens, Georgia, ES Cell International, Melbourne, Australia, Geron Corporation, Menlo Park, California, Göteborg University, Göteborg, Sweden, Karolinska Institute, Sweden, Maria Biotech Co. Ltd.
  • a stem cell for use in disclosed methods is a stem cell of neural or neuroendocrine origin, such as a stem cell from the central nervous system (see, for example US Patent Nos. 6,468,794 ; 6,040,180 ; 5,753,506 ; 5,766,948 ), the olfactory bulb or peripheral neural tissues (see, for example, Published US Patent Applications 20030003574 ; 20020123143 ; 20020016002 and Gritti et al. 2002 J Neurosci 22(2):437-45 ), the spinal cord (see, for example, US Patent Nos.
  • a neural stem cell is obtained from a peripheral tissue or an easily healed tissue, thereby providing an autologous population of cells for transplant.
  • Hematopoietic or mesenchymal stem cells may be employed in certain disclosed methods. Recent studies suggest that bone marrow-derived hematopoietic (HSCs) and mesenchymal stem cells (MSCs), which are readily isolated, have a broader differentiation potential than previously recognized. Purified HSCs not only give rise to all cells in blood, but can also develop into cells normally derived from endoderm, like hepatocytes ( Krause et al., 2001, Cell 105: 369-77 ; Lagasse et al., 2000 Nat Med 6: 1229-34 ). Similarly, HSCs from peripheral blood and from umbilical cord blood are expected to provide a useful spectrum of developmental potential.
  • HSCs bone marrow-derived hematopoietic
  • MSCs mesenchymal stem cells
  • MSCs appear to be similarly multipotent, producing progeny that can, for example, express neural cell markers ( Pittenger et al., 1999 Science 284: 143-7 ; Zhao et al., 2002 Exp Neurol 174: 11-20 ).
  • Examples of hematopoietic stem cells include those described in US Patent Nos. 4,714,680 ; 5,061,620 ; 5,437,994 ; 5,914,108 ; 5,925,567 ; 5,763,197 ; 5,750,397 ; 5,716,827 ; 5,643,741 ; 5,061,620 .
  • mesenchymal stem cells include those described in US Patent Nos.
  • Stem cell lines are preferably derived from mammals, such as rodents (e.g. mouse or rat), primates (e.g. monkeys, chimpanzees or humans), pigs, and ruminants (e.g. cows, sheep and goats), and particularly from humans, wherein the stem cell line is not derived from human embryonic stem cells.
  • stem cells are derived from an autologous source or an HLA-type matched source.
  • stem cells may be obtained from a subject in need of pancreatic hormone-producing cells (e.g. diabetic patients in need of insulin-producing cells) and cultured to generate autologous insulin-producing cells.
  • Other sources of stem cells are easily obtained from a subject, such as stem cells from muscle tissue, stem cells from skin (dermis or epidermis) and stem cells from fat.
  • cells for administration to a human should be compliant with good tissue practice guidelines set by the U.S. Food and Drug Administration (FDA) or equivalent regulatory agency in another country.
  • Methods to develop such a cell line may include donor testing, and avoidance of exposure to non-human cells and products.
  • Cells derived from a donor may be administered as unfractionated or fractionated cells, as dictated by the purpose of the cells to be delivered.
  • Cells may be fractionated to enrich for certain cell types prior to administration. Methods of fractionation are well known in the art, and generally involve both positive selection (i.e., retention of cells based on a particular property) and negative selection (i. e., elimination of cells based on a particular property).
  • positive selection i.e., retention of cells based on a particular property
  • negative selection i. e., elimination of cells based on a particular property.
  • the particular properties e.g., surface markers
  • Methods used for selection/enrichment of cells may include immunoaffinity technology or density centrifugation methods.
  • Immunoaffinity technology may take a variety of forms, as is well known in the art, but generally utilizes an antibody or antibody derivative in combination with some type of segregation technology.
  • the segregation technology generally results in physical segregation of cells bound by the antibody and cells not bound by the antibody, although in some instances the segregation technology which kills the cells bound by the antibody may be used for negative selection.
  • any suitable immunoaffinity technology may be utilized for selection/enrichment of the selected cells to be used, including fluorescence-activated cell sorting (FACS), panning, immunomagnetic separation, immunoaffinity chromatography, antibody-mediated complement fixation, immunotoxin, density gradient segregation, and the like.
  • FACS fluorescence-activated cell sorting
  • the desired cells the cells bound by the immunoaffinity reagent in the case of positive selection, and cells not bound by the immunoaffinity reagent in the case of negative selection
  • Immunoaffinity selection/enrichment is typically carried out by incubating a preparation of cells comprising the desired cell type with an antibody or antibody-derived affinity reagent (e.g., an antibody specific for a given surface marker), then utilizing the bound affinity reagent to select either for or against the cells to which the antibody is bound.
  • the selection process generally involves a physical separation, such as can be accomplished by directing droplets containing single cells into different containers depending on the presence or absence of bound affinity reagent (FACS), by utilizing an antibody bound (directly or indirectly) to a solid phase substrate (panning, immunoaffinity chromatography), or by utilizing a magnetic field to collect the cells which are bound to magnetic droplets via the affinity reagent (immunomagnetic separation).
  • undesirable cells may be eliminated from the preparation using an affinity reagent which directs a cytotoxic insult to the cells bound by the affinity reagent.
  • the cytotoxic insult may be activated by the affinity reagent (e.g., complement fixation), or may be localized to the target cells by the affinity reagent (e.g., immunotoxin, such as ricin B chain).
  • cells may be labeled at a desired step during the preparation for administration to the patient.
  • Imaging reagent A variety of methods may be used to label cells with imaging reagent.
  • cells will be placed in contact with imaging reagent such that the imaging reagent becomes associated with the cell.
  • Conditions will often be standard cell culture conditions designed to maintain cell viability.
  • the term "associated" is intended to encompass any manner by which the imaging reagent and cell remain in sufficiently close physical proximity for a sufficient amount of time as to allow the imaging reagent to provide useful information about the position of the cell, whether in vivo or in vitro.
  • Imaging reagent may be located intracellularly, e.g. after phagocytosis or surfactant mediated entry into the cell.
  • Immune cells such as dendritic cells, macrophages and T cells are often highly phagocytic and data presented herein and in other studies demonstrate that such cells, and other phagocytic cell types, are readily labeled. Other cell types, such as stem cells may also be labeled, regardless of phagocytic activity.
  • Imaging reagent may be inserted into a cell membrane or covalently or non-covalently bound to an extracellular component of the cell. For example, certain linear fluorocarbons described herein may be derivatized to attach one or more targeting moiety. A targeting moiety will be selected to facilitate association of the imaging reagent with the cell to be labeled.
  • a targeting moiety may be designed to cause non-specific insertion of the fluorocarbon into a cell membrane (e.g., a hydrophobic amino acid sequence or other hydrophobic moiety such as a palmitoyl moiety or myristoyl moiety) or to facilitate non-specific entry into the cell.
  • a targeting moiety may bind to a cell surface component, as in the case of receptor ligands.
  • a targeting moiety may be a member of a specific binding pair, where the partner is a cell surface component.
  • the targeting moiety may be, for example, a ligand for a receptor, or an antibody, such as a monoclonal or polyclonal antibody or any of the various polypeptide binding agents comprising a variable portion of an immunoglobulin (e.g., Fv fragment, single chain Fv (scFv) fragment, Fab' fragment, F(ab')2 fragment, single domain antibody, camelized antibody, humanized antibody, diabodies, tribodies, tetrabodies).
  • the fluorocarbon imaging reagent comprises perfluoro-15-crown ether.
  • transfection agents consist of cationic lipids, cationic liposomes, poly-cations, and the like.
  • the transfection agent is premixed with the fluorocarbon emulsion labeling agent, whereby it becomes associated with, or coats, the emulsion droplets.
  • the transfection agent-treated emulsion droplets are then added to the cultured cells and incubated so that the cells become labeled.
  • Common transfection agents include Lipofectamine (Invitrogen, Inc) FuGene, DOTAP (Roche Diagnostics, Inc.), and poly-L-lysine.
  • Protamines the major DNA-binding proteins in the nucleus of sperm in most vertebrates, package the DNA in a volume less than 5% of a somatic cell nucleus.
  • Protamines are simple proteins of low molecular weight that are rich in arginine and strongly basic.
  • Commercially available protamines come from the sperm of salmon and certain other species of fish.
  • the term "protamine” as used herein, refers to a low molecular weight cationic, arginine-rich polypeptide.
  • the protamine molecule typically comprises about 20 to about 200 amino acids and is generally characterized by containing at least 20%, 50% or 70% arginine.
  • Protamines are often formulated as salts, with one or more counter ions such as sulfate, phosphate and chloride.
  • protamines e.g., protamine sulfate
  • Suitable protamine sulfates can come from a variety of sources (e.g., salmon, herring, trout, etc.) and be of various grades and forms (e.g., USP, grades II, III, X, etc.), with and without histones or any recombinant derivative.
  • protamine solutions examples include protamine phosphate, protamine chloride, protamine sulfate-2, protamine sulfate-3, protamine sulfate-10, and protamine free base.
  • Data provided in this application shows self-deliverable nanoemulsions prepared with fluorocarbon imaging reagents (e.g., perfluoro-15-crown-5 ether or PFPE oxide) and incorporate a PluronicTM surfactant, optionally with Protamine Sulfate, or Cremophor® EL with an emulsifier and an additive.
  • fluorocarbon imaging reagents e.g., perfluoro-15-crown-5 ether or PFPE oxide
  • PluronicTM surfactant optionally with Protamine Sulfate, or Cremophor® EL
  • Simple co-incubation of cells with certain self-deliverable nanoemulsions provides sufficient cell labeling for imaging, without the need for transfection reagents.
  • Labeled cells may be monitored regardless of whether the cells are delivered directly to a particular site or delivered systemically.
  • labeled DCs were successfully imaged following either a focal implantation directly into tissues or an intravenous injection, and T-cells were imaged following intraperitoneal injection.
  • Cells may be inserted into a delivery device which facilitates introduction by injection or implantation into the subjects.
  • delivery devices may include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject.
  • the tubes additionally have a needle, e.g., a syringe, through which the cells of the disclosure can be introduced into the subject at a desired location.
  • the cells may be prepared for delivery in a variety of different forms.
  • the cells may be suspended in a solution or gel or embedded in a support matrix when contained in such a delivery device.
  • Cells may be mixed with a pharmaceutically acceptable carrier or diluent in which the cells of the disclosure remain viable.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
  • the solution is preferably sterile and fluid.
  • the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Solutions of the disclosure may be prepared by incorporating cells as described herein in a pharmaceutically acceptable carrier or diluent and, as required, other ingredients enumerated above, followed by filtered sterilization.
  • nuclear magnetic resonance techniques may be used to detect populations of labeled cells.
  • the term “detect” is used to include any effort to ascertain the presence or absence of a labeled molecule or cell, particularly by a nuclear magnetic resonance technique.
  • the term “detect” is also intended to include more sophisticated measurements, including quantitative measurements and two- or three-dimensional image generation.
  • MRI may be used to generate images of such cells.
  • the labeled cells may be administered to a living subject. Following administration of the cells, some portion of the subject, or the entire subject, may be examined by MRI to generate an MRI data set. In other instances, the emulsion is injected directly iv, and the subject is subsequently imaged at one or more time points.
  • a "data set”, as the term is used herein, is intended to include raw data gathered during magnetic resonance probing of the subject material, the acquisition parameters, as well as information processed, transformed or extracted from the raw data.
  • the raw data includes transient signals obtained by MRI/MRS, including the free-induction decays, spin-echoes, stimulated-echoes, and/or gradient echoes.
  • Examples of processed information include two-dimensional or three-dimensional pictorial representations of the subject material.
  • the processed information may also include magnitude images, the real and imaginary image components, as well as the associated phase map images.
  • Another example of extracted information is a score representing the amount or concentration of imaging reagent or 19 F signal in the subject material.
  • the amount of 19 F signal in the subject material can be represented or calculated in many ways; for example, the average signal-to-noise-ratio (SNR) of the 19 F signal for a region of interest (ROI) may be measured and used to calculate the abundance of labeled cells.
  • SNR signal-to-noise-ratio
  • ROI region of interest
  • the average intensity, or pixel- or voxel-wise summation of the 19 F signal may be used to calculate the abundance of labeled cells.
  • Labeled cells may be examined in contexts other than in the subject. It may be desirable to examine labeled cells in culture. In certain embodiments, labeled cells may be applied to or generated within a tissue sample or tissue culture, and labeled cells may therefore be imaged in those contexts as well. For example, an organ, tissue or other cellular material to be transplanted may be contacted with an imaging reagent to generate labeled cells prior to implantation of such transplant in a subject.
  • labeling agents of the disclosure are designed for use in conventional MRI detection systems.
  • MRI magnetic resonance imaging
  • 19 F an alternate nucleus is detected, 19 F.
  • 19 F MRI has only slightly less intrinsic sensitivity compared to 1 H; the relative sensitivity is approximately 0.83. Both have a nuclear spin of +1/2.
  • the natural isotopic abundance of 19 F is 100%, which is comparable to 99.985% for 1 H.
  • the physical principles behind the detection and image formation are the same for both 1 H and 19 F MRI.
  • the subject material is placed in a large static magnetic field.
  • the field tends to align the magnetic moment associated with the 1 H or 19 F nuclei along the field direction.
  • the nuclei are perturbed from equilibrium by pulsed radio-frequency (RF) radiation at the Larmor frequency, which is a characteristic frequency proportional to the magnetic field strength where nuclei resonantly absorb energy.
  • RF radio-frequency
  • the nuclei Upon removing the RF, the nuclei induce a transient voltage in a receiver antenna; this transient voltage constitutes the nuclear magnetic resonance (NMR) signal.
  • Spatial information is encoded in both the frequency and/or phase of the NMR signal by selective application of magnetic field gradients that are superimposed onto the large static field.
  • the transient voltages are generally digitized, and then these signals may be processed by, for example, using a computer to yield images.
  • the Larmor frequency of 19 F is only slightly lower ( ⁇ 6 %) compared to 1 H.
  • the 19 F detection may be coupled with different types of magnetic resonance scans, such as MRI, MRS or other techniques.
  • MRI magnetic resonance scans
  • MRS magnetic resonance scans
  • the proton MRI will provide an image of the subject material and allow one to define the anatomical context of the labeled cells detected in the 19 F image.
  • data is collected for both 19 F and 1 H during the same session; the subject is not moved during these acquisitions to better ensure that the two data sets are in spatial registration.
  • 19 F and 1 H data sets are acquired sequentially, in either order.
  • An RF coil i.e. antenna
  • Tuning between these two frequencies can be performed manually (e.g. via an electro-mechanical variable capacitor or inductor), or electrically, via active electronic circuitry.
  • both data sets can be acquired simultaneously, for example, to conserve imaging time.
  • Simultaneous acquisition of the 19 F and 1 H data sets require an RF coil or antenna that can be electrically tuned simultaneously to the 19 F and 1 H Larmor frequency (i.e., a double-tuned coil).
  • the RF coil can be "broadband," with one broadly-tuned electrical resonance that covers both Larmor frequencies (i.e. 19 F and 1 H).
  • Other imaging techniques, such as fluorescence detection may be coupled with 19 F MRI. This will be particularly desirable where a fluorocarbon imaging reagent has been derivatized with a fluorescent moiety.
  • the 19 F MRI scan may be combined with a PET scan in the same subject or patient by using dual-model radioactive 18 F/ 19 F fluorocarbon labeling reagents as described herein.
  • MRI examination may be conducted according to any suitable methodology known in the art.
  • Many different types of MRI pulse sequences, or the set of instructions used by the MRI apparatus to orchestrate data collection, and signal processing techniques e.g. Fourier transform and projection reconstruction
  • signal processing techniques e.g. Fourier transform and projection reconstruction
  • the reagents and methods of this disclosure are not tied to any particular imaging pulse sequence or processing method of the raw NMR signals.
  • MRI methods that can be applied to this disclosure broadly encompasses spin-echo, stimulated-echo. gradient-echo, free-induction decay based imaging, and any combination thereof.
  • Fast imaging techniques where more than one line in k-space or large segments of k-space are acquired from each excited signal, are also highly suitable to acquire the 19 F (or 1 H) data.
  • fast imaging techniques include fast spin-echo approaches (e.g. FSE, turbo SE, TSE, RARE, or HASTE), echo-planar imaging (EPI), combined gradient-echo and spin-echo techniques (e.g. GRASE), spiral imaging, and burst imaging.
  • MRS can be used to detect the presence of fluorocarbon-labeled cells in localized tissues or organs.
  • MRS methods are implemented on a conventional MRI scanner.
  • VOI volume of interest
  • the magnitude of the 19 F NMR signal observed within the VOI is directly related to the number of labeled cells, and/or the mean concentration of PFPE per cell present in the tissue or organ.
  • Methods for isolating a VOI within a much larger subject are well known the art (for example, Magnetic Resonance Imaging, Third Edition, Chapter 9, Editors D.D. Stark and W.G.
  • Examples include using a localized RF surface coil near the VOI, surface spoiling, surface coil B 1 -gradient methods, slice-selective B 0 -gradient techniques, STEAM, PRESS, image selective in vivo spectroscopy (ISIS), and magnetic resonance spectroscopic imaging (MRSI).
  • ISIS image selective in vivo spectroscopy
  • MRSI magnetic resonance spectroscopic imaging
  • the subject material is a fixed or otherwise preserved specimen of tissue that has been biopsied or necropsied from the animal or human.
  • the subject material is then subjected to conventional high-resolution, one or multi-dimensional, liquid state 19 F NMR to determine the amount of fluorine present in the sample.
  • the fluorine content is directly related to the number of labeled cells in the subject materal specimen.
  • the amount of 19 F measured in the sample is directly proportional to the number of these phagocytes present in the tissue.
  • 19 F NMR to analyze the 19 F content of the tissue, one uses one-dimension 19 F NMR.
  • a 19 F reference compound will be added to the sample of known number of 19 F spins that has a chemical shift that is different than the composition of the cell labeling emulsion (see below).
  • the relative integrated areas under the emulsion peak and reference peak can be used to calculate the absolute number of fluorines present in the tissue sample.
  • the weight of the tissue sample can also be incorporated into this calculation to extract the mean fluorine density of the tissue sample, and this parameter can be considered a quantitative index of inflammation or "inflammation index".
  • An ROI may include all labeled cells in a subject or labeled cells in specific organs such as the pancreas, specific tissues such as lymph nodes, or any region or of one or more voxels showing detectable MRI/MRS 19 F signal.
  • a ROI can be an otherwise undefined area beyond a particular experiment.
  • calibrating the mean "cellular dose" of 19 F labeling agent pre-implantation of a particular cell population is often a pre-requisite for quantitative cell determinations in subject materials or the patient. It is anticipated that different cell types have different innate abilities to take up the labeling agents in vitro, and thus the cellular dose of the labeling agent will also vary. Furthermore, different cells of the same type acquired from different sources (e.g., different patients) may have different affinities for the labeling agent. Thus a cellular dose calibration may be required.
  • This calibration may be used, initially, to modify the labeling protocol (i.e., incubation conditions, duration of time that cells are incubated with labeling fluorocarbon emulsion, concentration of fluorocarbon emulsion in culture medium during labeling, etc.) to achieve a certain range of cellular dose before labeled cells are actually used in a subject to be imaged.
  • the labeling protocol i.e., incubation conditions, duration of time that cells are incubated with labeling fluorocarbon emulsion, concentration of fluorocarbon emulsion in culture medium during labeling, etc.
  • the mean value 19 F labeled per cell as is, for subsequent quantification in the subject to be imaged.
  • the mean number of 19 F molecules (F's) per cell of a labeled cell population is measured (i.e., calibrated) in vitro prior to administration of the cells to the subject or patient.
  • the mean number of 19 F molecules (F's) per cell of a labeled cell population is calibrated in a test population of cells of a particular type, not necessarily destined for a patient, but used to calibrate cellular dose of labeling agent as a consequence of a particular labeling protocol or set of conditions; optionally, the value of cellular dose is then used for future labeling and in vivo imaging experiments in the same population type of cells with the same labeling protocol.
  • the cellular dose of labeling agent can be assayed in vitro using a variety of quantitative techniques. For example, one can use a one-dimensional (1D) 19 F NMR spectrum obtained from a cell pellet, cell suspension, or cell lysate, of a known number of labeled cells. From this spectrum, one can calculate the integrated area of the 19 F spectrum or a portion thereof, originating from the labeling reagent associated with the cells.
  • the integrated area of the 19 F spectrum, denoted S cells is directly proportional to the total amount of 19 F in the cell pellet, suspension, or lysate.
  • the measured S cells may be normalized to a 19 F standard.
  • a 19 F standard can be, for example, a solution of a known volume and concentration of a fluoro-chemical, where one can calculate the total number of 19 F nuclei in the standard, denoted F stan .
  • a suitable fluoro-chemical reference ideally has a simple 19 F NMR spectrum, preferable with a single narrow resonance (e.g. trifluoroacetic acid or TFA) and optionally a 19 F chemical shift that is significantly different than the labeling fluorocarbon.
  • the 19 F standard can be placed in the same NMR tube as the labeled cell material being measured, in a separate tube, or optionally can be measured in a separate experiment using the same NMR instrument.
  • the integrated area of the spectrum from the 19 F standard, denoted S stan can then be measured.
  • the mean number of 19 F per labeled cell denoted F c
  • F c S cells S s tan F s tan 1 N cells where N cells is the number of labeled cells contained in the in vitro test sample.
  • Quantitative NMR methods for 19 F and other nuclei are well know in the art, and those skilled can devise many variations to the cellular dose calibration procedure described above.
  • 19 F NMR there are other quantitative methods that can be used to assay the cellular dose of the labeling reagent.
  • a reagent may be labeled fluorescently, luminescently, optically, or radioactively (see US Patent Application No. 2007-0258886 ).
  • in situ cell labeling of circulating phagocytes following iv injection of emulsion to meaure the effective cell labeling, one can extravesate a portion of peripheral blood from the subject and measure the effective cell loading of leukocytes using the methods described above.
  • one or more of the various cell sorting or enrichment techniques can be used to sort out phagocytic cells (e.g., macrophages) prior to the loading measurement (above) to better define which cell population has been labeled in situ.
  • the measured cell labeling parameter can then be used to calculate the apparent number of inflammatory cells present in tissue using the magnetic resonance methods described herein.
  • a calibrated external 19 F reference i.e. phantom
  • the image intensity of the calibrated phantom is used, for examples, when analyzing the 19 F MRI/MRS data set to proved an absolute standard for the number of 19 F nuclei when examining the subject material or patient.
  • the calibrated phantom is used to normalize the sensitivity of the particular MRI/MRS system that has been loaded with a particular subject to be imaged.
  • the 19 F reference may be, for example, one or more vessels containing a solution of a known concentration of 19 F nuclei.
  • the solution contains a dilute concentration of the emulsified fluorocarbon labeling reagent.
  • the solution contains non-emulsified fluorocarbon labeling reagent, a gel, or liquid, for example that has been diluted in a suitable solvent.
  • the solution can be comprised of another fluoro-chemical, ideally with a simple 19 F NMR spectrum, preferable with a single narrow NMR resonance (e.g.
  • the T1 and T2 values of the reference solution are similar to those of the labeling reagent.
  • the solution can contain perfluorocarbon-labeled cells, or lysates of the same.
  • the non-cellular reference has the advantage of longer storage times.
  • the solution can take the form of a gel.
  • the vessel containing the solution is preferably sealable, and can take a variety of geometries; preferred vessel geometries include ellipsoidal, cylindrical, spherical, and parallel piped shapes.
  • One or more vessels containing 19 F reference solution can be used during the 19 F MRI/MRS of the subject material. If multiple 19 F references (i.e. vessels) are used they can contain the same 19 F concentration or different concentrations, and in the case of the latter, they ideally contain graded concentrations of fluorochemical.
  • the placement of the calibrated 19 F reference vessel(s) can be placed preferably externally or alongside, or optionally inside, the imaged subject or patient prior to data acquisition.
  • the reference is imaged using 19 F MRI along with the subject in the same image field of view (FOV).
  • 19 F MRS data is acquired in the reference either sequentially or in parallel with the subject data set.
  • data from the reference can be acquired using MRI/MRS acquired in a separate scan.
  • the external reference is not scanned along with a subject in every 19 F MRI/MRS examination, but rather, values of the reference 19 F signal intensity acquired using MRI/MRS is used from a scan of a comparable subject or a simulated-subject.
  • the calibrated 19 F standard may be sampled by one or more voxels.
  • the observable 19 F intensity produced by a voxel may be proportional to the concentration of the fluorochemical in the solution (or gel) and the voxel volume.
  • the reference standard is comprised of many voxels.
  • the mean intensity of one, several, or all voxels in the reference standard is calculated over an ROI defined within the 19 F image of the reference standard.
  • the physical geometry of the reference standard vessel contributes to defining the observed 19 F signal intensity; for example, the volume compartment(s) containing the 19 F reference solution is smaller than the voxel volume.
  • the calibrated external reference relies on a solution with a 1 H signal intensity of a known number of detectable 1 H; in this case the sensitivity of the 19 F signal in the subject material is reference to a 1 H calibrated standard.
  • the solution or gel in the 1 H calibrated reference yields a simple 1 H NMR spectrum, preferable with a single narrow NMR resonance (e.g., H 2 O, or mixtures of H 2 O-D 2 O).
  • the use of the 1 H standard reference is the same in many other respects as described above for the 19 F reference.
  • the calibrated reference standard contains any other MRI/MRS-active nuclei.
  • the reference is an internal organ or tissue detected via 1 H MRI/MRS, where the data may be raw or normalized.
  • the reference is a standard that is not scanned with the subject, but is calibrated by relevant factors such as the weight of the patient or the size of the body cavity.
  • a key set of parameters may include: (i) the cellular dose of labeling agent (i.e., F c ) measured in vitro; (ii) in vivo 19 F MRI/MRS data set taken in the subject at one or more time points following labeled cell administration; (iii) the voxel volume; (iv) the in-plane voxel area (i.e., area of the image pixel); (v) optionally, the MRI/MRS data set from the 19 F reference standard; (vi) optionally, the measured Johnson noise of the 19 F MRI/MRS data in the subject material; (vii) optionally, the measured signal-to-noise ratio (SNR) of one or more voxels of the 19 F MRI/MRS data set in the subject material;
  • SNR signal-to-noise ratio
  • N c the effective number of labeled cells seen by 19 F MRI in the subject material
  • N c total number of labeled cells in the ROI
  • [F R ] concentration of 19 F in the calibrated 19 F reference solution (or gel)
  • v voxel volume
  • I R mean intensity of the calibrated 19 F reference taken with the MRI/MRS scan, averaged over one or more voxels
  • F c average 19 F cellular dose of the labeling agent measured in vitro
  • N ROI number of voxels in the ROI containing labeled cells
  • I c (i) image intensity of the i th voxel in the ROI containing labeled cells
  • i unitless index for voxels in the ROI containing labeled cells.
  • N c is the average intensity of the ROI containing the labeled cells, (i.e. the average intensity of the N ROI voxels).
  • N c ⁇ I c avg I R V c 1 F c F R where V c is the total volume of the ROI containing the labeled cells.
  • N c ⁇ I c avg I R V c V R 1 F c N R where V R is the effective volume of the reference in the 19 F MRI/MRS and N R is the number 19 F nuclei in V R .
  • V R is the effective volume of the reference in the 19 F MRI/MRS
  • N R is the number 19 F nuclei in V R .
  • N c there are many ways to estimate the number of labeled cells, N c , and many similar forms of these basic expressions can be derived by basic mathematical manipulations, however, all rely on the same basic content contained within the input parameters described by (i-x). Furthermore, quantification of labeled cells in an ROI need not be expressed in terms of absolute numbers or effective cell numbers. Other quantitative indices can be derived that are indicative of the amount of cells in an ROI. For example, one can calculate the ratio I c avg /I R , or the ratio of the average SNR values observed in the ROI and the reference; all of these fall within subsets of the above expressions and/or the parameters.
  • the 19 F MRI data set of the subject material can undergo post-processing before the actual cell quantification calculation is performed (as described above).
  • post-processing algorithms may include "de-noising" the 19 F data set. This can be accomplished by, for example, by thresholding the image to cut off low-intensity noise; this involves rescaling the image intensity so that low values are set to zero. In magnitude MRI images, random Johnson noise is often apparent and uniformly distributed across the image FOV. It is well know in the art that one can threshold out the low-level image intensity so that regions known to contain no true signal (i.e. devoid of 19 F and/or 1 H nuclei) appear to have a null or very near-null intensity.
  • de-noising of the data set can be achieved by using other algorithms, for example using wavelet analysis, and many methods are known in the art for image de-noising: Khare, A., et al., INTERNATIONAL JOURNAL OF WAVELETS MULTIRESOLUTION AND INFORMATION PROCESSING, 3 (4): 477-496 DEC 2005 ; Cruz-Enriquez, H., et al., IMAGE ANALYSIS AND RECOGNITION, 3656: 247-254 2005 ; Awate, SP., et al., INFORMATION PROCESSING IN MEDICAL IMAGING, PROCEEDINGS, 3565: 677-688 2005 ; Ganesan, R., et al., IIE TRANSACTIONS, 36 (9): 787-806 SEP 2004 ; Scheunders, P., IEEE TRANSFER
  • the above set of key parameters (i-x) can be used to derive quantitative or statistical measures of the accuracy or confidence of the measured number of labeled cells or related indices.
  • 19 F MRI/MRS data sets are often subject to SNR limitations within ROI, and thus it is often useful to calculate a metric of the confidence or accuracy of the measurement.
  • Many methods are known in the art for the statistical analysis of MRI and other biomedical-type images. The claimed embodiment is understood to encompass these known methods.
  • the emulsions of the application can be administered in a variety of unit dosage forms.
  • the dose will vary according to the particular emulsion.
  • the dose will also vary depending on the manner of administration, the overall health, condition, size, and age of the patient.
  • administration of the emulsions may be performed by an intravascular route, e.g., via intravenous infusion by injection.
  • other routes of administration may be used.
  • Formulations suitable for injection are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985 ). Such formulations must be sterile and non-pyrogenic, and generally will include a pharmaceutically effective carrier, such as saline, buffered (e.g., phosphate buffered) saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions, and the like.
  • the formulations may contain pharmaceutically acceptable auxiliary substances as required, such as, tonicity adjusting agents, wetting agents, bactericidal agents, preservatives, stabilizers, and the like.
  • suitable buffers for intravenous administration are used to aid in emulsion stability.
  • glycols are used to aid in emulsion stability.
  • administration of the emulsions may be performed by a parenteral route, typically via injection such as intra-articular or intravascular injection (e.g., intravenous infusion) or intramuscular injection.
  • parenteral route typically via injection such as intra-articular or intravascular injection (e.g., intravenous infusion) or intramuscular injection.
  • Other routes of administration e.g., oral (p.o.), may be used if desired and practicable for the particular emulsion to be administered.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions of the application.
  • formulations of the subject emulsions are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances.
  • Endotoxins include toxins that are confined inside microorganisms and are released when the microorganisms are broken down or die.
  • Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions.
  • FDA Food & Drug Administration
  • EU endotoxin units
  • Formulations of the subject emulsions include those suitable for oral, dietary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), ophthalmologic (e.g., topical or intraocular), inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops), rectal, and/or intravaginal administration.
  • parenteral e.g., intravenous, intraarterial, intramuscular, subcutaneous injection
  • ophthalmologic e.g., topical or intraocular
  • inhalation e.g., intrabronchial, intranasal or oral inhalation, intranasal drops
  • rectal e.g., rectal, and/or intravaginal administration.
  • Other suitable methods of administration can also include rechargeable or biodegradable devices and controlled release polymeric devices.
  • Stents in particular, may be coated with a controlled release polymer
  • the amount of the formulation which will be therapeutically effective can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the dosage of the compositions to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.
  • Therapeutics of the disclosure can be administered in a variety of unit dosage forms and their dosages will vary with the size, potency, and in vivo half-life of the particular therapeutic being administered.
  • emulsions may be formulated to have optimal pharmacokinetic properties to enable uptake by phagocytes before clearance of the emulsion.
  • Doses of therapeutics of the disclosure will also vary depending on the manner of administration, the particular use of the emulstion, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician.
  • the formulations of the application can be distributed as articles of manufacture comprising packaging material and a pharmaceutical agent which comprises the emulsion and a pharmaceutically acceptable carrier as appropriate to the mode of administration.
  • the pharmaceutical formulations and uses of the disclosure may be combined with any known compositions for the applications of the application.
  • Methods for quantifying labeled cells will typically be conducted with the aid of a computer, which may operate software designed for the purpose of such quantification.
  • software may be a stand-alone program or it may be incorporated into other software, such as MRI image processing software (see US Patent Application No. 2007-0253910 ).
  • nanoemulsions are prepared with Perfluoro-15-crown ether, a well known fluorocarbon in the art used for formulating emulsions as blood substitutes, for oxygen sensing and cell tracking (see US Patent Nos. 4,838,274 and 5,785,950 ).
  • Perfluoro-15-crown ether a well known fluorocarbon in the art used for formulating emulsions as blood substitutes, for oxygen sensing and cell tracking (see US Patent Nos. 4,838,274 and 5,785,950 ).
  • novel approaches to formulate stable nanoemulsions with perfluoro-15-crown 5 ether we introduced novel approaches to formulate stable nanoemulsions with perfluoro-15-crown 5 ether.
  • Other novel formulations incorporate various co-emulsifiers rendering the emulsions "self deliverable" into various cell types.
  • novel emulsion formulations described herein show marked improvments over prior art emulsions, particularly with respect to a decreased nanoemulsion droplet size (as low as 110 nm), which is advantageous for in vivo applications.
  • Presented nanoemulsions presented are prepared as monodisperse (PDI ⁇ 0.1) and show exceptional stability in presence of serum and at body temperatures. Further novelty lies in introducing protamine sulfate or other co-surfactants for achieving "self-deliverable" properties and the use of Cremophor EL, for achieving exceptionally small droplet size and high stability in vivo.
  • nanoemulsions were prepared on 0.5-1 liter scale using microfluidization.
  • the emulsions were analyzed by dynamic light scattering (DLS) for droplet size and polydispersity using a Malvern Zetasizer Nano ZS. The appearance of the emulsions was evaluated visually.
  • the nanoemulsions were tested for serum stability, pH and osmolality. Furthermore, the efficacy for in vivo experiments has been demonstrated.
  • Formulation perfluoro-15-crown-5-ether % w/w Emulsifier 1 % w/w Emulsifier2 % w/w 1 15 % 0.6 % Pluronic F68 2 15 % 0.6 % Pluronic F68 0.04 %
  • a highly concentrated pre-emulsion was prepared first, diluted with sterile water to reach needed final concentration and finally processed by MF to obtain an emulsion with an acceptable droplet size ( ⁇ 200 nm) and polydispersity ( ⁇ 0.15). Emulsifiers and additives were dissolved in sterile water right before use. Pluronic F68 solution was prepared in water at 100 mg/ml and protamine sulfate at 20 mg/mL. The concentrated pre-emulsion was prepared by processing all the required liquids (perfluoro-15-crown 5 ether oil, F68 solution and Protamine Sulfate solution) with rotary shear (using an ultra-turrax shaft with a diameter of 25mm) at 13500 rpm for 2.5 minutes.
  • This first mixture was then diluted to the final needed concentration and reprocessed with rotary shear for 1 minute.
  • the pre-emulsions were immediately processed by MF, using an M-110S microfluidizer (Microfluidics Corp.).
  • the liquid pressure during microfluidization processing was >18500 psi, and the low droplet size was achived by 5 to 8 discrete passes (cycles).
  • the nanoemulsion was sterilized by filtration.
  • the product was filtered using a 47 mm PFR (PTFE, 0.22 ⁇ m) disc in a filter holder (PALL, Inc.). Succesful filtration was achieved using low flow of 2-8ml/min.
  • Emulsions were visually inspected and subjected to droplet size and polydisperisy measurements by DLS. All samples were diluted with WFI to reach final concentration of 1% perfluoro-15-crown-5-ether prior to DLS measurements.
  • the droplet size was ⁇ 200 nm and polydispersity ⁇ 0.2 for 3 months upon storage at 5°C and room temperature.
  • Cremophor® EL is a non-ionic solubilizer where the main component of is glycerol-polyethylene glycol ricinoleate, which, together with fatty acid esters of polyethyleneglycol, represents the hydrophobic part; the smaller, hydrophilic component consists of polyethylene glycols and ethoxylated glycerol.
  • the lipid component incorporates into the liposomal coat of the nanoemulsion droplet, while the PEG secures steric stabilization.
  • the resulting nanoemulsion droplet has a perfluoro-15-crown-5-ether core, a liposomal coating, and a sterically stabilized surface via the PEG portion of Cremophor EL.
  • Steric stabilization improved markedly shelf life and nanoemulsion stability in vivo.
  • the prior art formulation emulsion 4 was prepared following a previously reported procedure ( WO2006096499 ) side-by-side with the new formulation (emulsion 3).
  • Emulsions compositions with medium of WFI and liposomes which consisted of 70 mol% lecithin, 28 mol% cholesterol, and 2 mol% DPPE.
  • Formulation perfluoro-15-crown-5-ether % w/v Emulsifier1 % w/v Emulsifier2 % w/v Additive % w/v 3 35.6% 3.0% Cremophor ELP 2.0% Liposomes 2.0% Propylene Glycol 4 35.6% 2.0% Safflower oil 2.0% Liposomes 1.7% Glycerin
  • liposomes were prepared by sonification of the liposomal components.
  • a concentrated pre-emulsion was prepared by adding perfluoro-15-crown-5-ether/emulsifier1 and additive; subsequently the batch was diluted with water for injection to the final concentration and processed using the MF to the final oil droplet size.
  • the liposomal components lecithin, cholesterol and DPPE were disolved in chloroform and dried by rotary evaporation into a film. The lipids were then dispersed in WFI by sonication. The resulting suspensions were flushed with argon (gas), closed and stored protected from light at 5°C until use. All components, including Cremophor® EL (or safflower oil), perfluoro-15-crown-5-ether, propylene glycol, and liposomes were first combined with small amount of water. This initial concentrated mixture had perfluoro-15-crown-5-ether at 60% w/w.
  • the mixture was processed by rotary sheer (turrax) for two minutes at 12500 rpm, and the resulting concentrated pre-emulsion further diluted with WFI to the final volume of and processed again for 1 minute at 12500 rpm.
  • the pre-emulsion was not stable and thus was immediately processed by microfluidization with 5 to 8 discreet passes (cycles) in the MF with a pressure of > 18500 psi.
  • the final emulsion product was stable as described below.
  • Formulation 5 was prepared using the procedure as described above for formulation 3 , wherein perfluoro-15-crown-5 ether was replaced by weight with linear PFPE. This replacement was feasible due to similar specific weight and viscosity between the linear PFPE and perfluoro-15-crown-5 ether.
  • the amount of PFPE oxide in formulation 5 was equivalent to weight amount of perfluoro-15-crown 5 ether in formulation 3, with the advantage of obtaining a larger number of equivalent 19F spins/nanoemulsion droplet.
  • the surfactant comixture, Cremophor EL and additive amounts were the same as those described for formulation 3.
  • Cremophor® EL or safflower oil
  • linear PFPE linear PFPE
  • propylene glycol propylene glycol
  • liposomes were first combined with a small amount of water.
  • This initial concentrated mixture comprised linear PFPE at 60% w/w.
  • the mixture was processed by rotary sheer (turrax) for two minutes at 12500 rpm, and the resulting concentrated pre-emulsion was further diluted with WFI to the final volume and processed again for 1 minute at 12500 rpm.
  • the pre-emulsion was stable and was immediately processed by microfluidization with 5 to 8 discreet passes (cycles) in the MF with a pressure of > 18500 psi.
  • Figure 7 shows droplet size measurements by DLS for formulation 5 at day 1 and day 342.
  • Emulsion 3 was followed by DLS and visually inspected for signs of destabilization for a total of > 6 months.
  • a summary of the stability data for formulation 3 is shown in Figure 1 .
  • Formulation 3 appeared upon visual inspection as turbid and milky; no large droplets, aggregates, sedimentation, or phase separation was observed during the follow up.
  • the droplet size of the formulation 3 emulsion was smaller as compared to all other emulsions tested, and most importantly the droplet size and PDI were dramatically decreased as compared to formulation 4 ( WO2006096499 ). Comparative results are shown in Table 4.
  • Introduction of Cremophor EL dramatically decreased the droplet size in formulation 3, as compared to formulation 4 prepared with safflower oil under the same manufacturing conditions.
  • Formulation 4 with safflower oil was prepared in parallel with the formulation 3 using previously reported methods ( WO2006096499 ).
  • the droplet size, PDI and serum stability of this emulsion was compared to the formulation 3, which utilizes Cremophor EL (above).
  • the droplet size of formulation 4 was substantionally larger than compared to the formulations 1, 2, or 3 (Tables 4 and 7).
  • Droplet size increased approximately 25% after only one week at 5 °C (Table 7). After a 2-week period the apparent plateau value for the droplet size was reached (Table 7).
  • Polydispersity increased considerably, which was not observed in the other formulations.
  • Table 8 shows the serum stability of formulation 4.
  • a fluorescent dye was incorporated into lipid layer of the formulation 3 post-processing.
  • This addition created a 'dual-modality' agent that can be detected both by 19 F magnetic resonance and various fluorescence methods (e.g., flow cytometry, histology, FACs analysis, fluorescence microscopy and the like).
  • Lipophilic dyes that are widely available commercially (e.g., dialkylcarbocyanines, Invitrogen, Inc.) were used.
  • DiI Molecular Probes
  • DiI Molecular Probes
  • the DiI was incorporated into the liposomal coating of formulation 3.
  • Nanoemulsions 3 and 5 may also be formulated with protamine sulfate and polyethylamine to improve uptake in non-phagocitic cells. These polyamines are incorporated into the pre-emulsion and integrated into the emulsion surfactant layer following MF processing, as described above. The amount of polyamine will be optimized to achieve optimal cell labeling. (See WO2009/009105 ).
  • Fluorescent blended PFPE amides have been recently described that contained covalently conjugated fluorescent dyes (e.g. BODIPyTR, FITC or Alexa647). These fluorescent conjugated PFPE oils behave as unique single fluorocarbon phase during nanoemulsion processing [for details see Janjic et al, J Am Chem Soc. 2008 Mar 5;130(9):2832-41 ]. Fluorescent versions of the formulations 3 and 5 may be prepared with FBPAs. In formulation 3, 10% v/v of the perfluoro-15-crown-5 ether can be replaced by FBPA. Consequently, in formulation 5, 10% v/v of PFPE oxide is replaced by FBPA.
  • FBPA Fluorescent blended PFPE amides
  • the oils are carefully blended together to obtain unique fluorocarbon phase, and then subjected to nanoemulsion preparation procedures as described for formulation 3 and formulation 5.
  • the advantage of fluorocarbon phase being labeled with fluorescent dye rather then the surfactant co-mixture is multifold.
  • the fluorescent dye in these new formulations remains within the fluorocarbon core of the nanoemulsion droplet throughout the processing, during cell labeling, and presumably in vivo.
  • the fluorescent signal is directly proportional to the 19 F NMR signal from the labeled tissue or cells [ Janjic et al, J Am Chem Soc. 2008 Mar 5;130(9):2832-41 ], thus there is no differential labeling beween cells (or tissues) for the two imaging or detection modalities (i.e., magnetic resonance and fluorescence).
  • Formulation 3 showed excellent stability and a very small droplet size ( ⁇ 150 nm) with low polydispersity ( ⁇ 0.15), as shown in Figure 1 .
  • the nanoemulsion is stable both in vitro, under cell culturing conditions, and in vivo, upon injection to rodents.
  • RAW cells ATCC, Manassass, VA
  • cytotoxicity was estimated by direct cell counts.
  • Minimal toxicity was observed at the highest dose applied and after 24 h exposure ( Figure 4A ).
  • Formulation 3 uptake in RAW cells was measured by 19 F NMR in cell pellets as describe above and showed clear dose dependence ( Figure 4B ).
  • Figure 5 shows a representative 19 F NMR spectrum of formulation 3 labeled RAW cells. Satisfactory uptake in phagocytic RAW cells was obtained after 18 h co-incubation and without transfection reagents.
  • protamine sulfate and polyethylamine were incorporated into formulation 3 as described in formulation 2 (above).
  • Formulation 3 cell labeling showed no impact on the cytokine producing capacity of RAW cells ( Figure 6 ).
  • the cells were activated by LPS for 24 h prior to being exposed to formulation 3 for 18 h, washed, and then cultured for 24 h.
  • An ELISA assay was used to measure cytokine production, including the levels of IL-6 and TNF-alpha. These tests showed that cells labeled with formulation 3 had no impact on their activity in vitro.
  • inflammation can be assayed in two ways - by using conventional high-resolution NMR or by using MRI. In both methods, one detects the abundance of 19 F nuclei in tissue, contained within phagocytic inflammatory cells (e.g., monocyte/macrophage/neutrophil). Most standard NMR instrumentation can routinely detect 19 F. NMR provides a sensitive and cost-effective approach for quantifying the degree of leukocyte infiltration in tissue samples. This approach abrogates the need for time-intensive pathological staining and subsequent cellular quantification via microscopy.
  • NMR nuclear magnetic resonance
  • formulation 3 was used to detect inflammation in a sponge granuloma model using in vivo MRI.
  • a PVS sponge disk was soaked in Complete Freund's Adjuvant (CFA) and subcutaneously implanted dorsally in a C57BL/6 mouse.
  • CFA Complete Freund's Adjuvant
  • a single intravenous injection of formulation 3 (0.5 mL) was given on day 4 post surgery.
  • the anesthetized mouse was imaged on day 5 at 7T.
  • Figure 9 shows a 1 H/ 19 F fusion image, with the 19 F rendered in pseudo-color.
  • the data shows an intense concentration of macrophages labeled with formulation 3 surrounding the sponge (asterisk).
  • formulation 3 was used to measure the inflammation profile in the spinal cord (SC) of a rodent model of multiple sclerosis, experimental allergic encephalomyelitis (EAE).
  • EAE experimental allergic encephalomyelitis
  • the EAE model was generated in a DA rat using a single subcutaneous inoculation in the tail base consisiting of isogenic spinal cord homogenate mixed with Complete Freund's Adjuvant (CFA).
  • CFA Complete Freund's Adjuvant
  • Clinical Stage-2 EAE rats were intravenously injected with formulation 3 (0.5 mL), and 48 hours later intact, fixed segments of the SC were assayed for inflammation using conventional 19 F NMR spectroscopy at 470 MHz ( Figure 10 ).
  • formulation 5 can be used to visualize acute and chronic inflammation in mouse models.
  • Klug et al. (Abstract #3172, Proc. Int. Soc. Mag. Reson. Med. 17, 2009 ) demonstrated that formulation 5 can be used to visualize acute and chronic inflammation in mouse models.
  • These investigators used both C57BL/6 mice, prepared with localized ear injections of TNF- ⁇ , and apoE -/- mice, a model of atherosclerotic plaques.
  • 19 F could be detected by MRI at sites of TNF- ⁇ injection in the C57BL/6 mice, and in the apoE -/- mice, 19 F could be detected in the brachiocephatic arch region, which is a common site of plaques in these animals.

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Claims (13)

  1. Wässrige Zusammensetzung umfassend Perfluor-15-krone-5-ether oder PFPE-oxid, einen Emulgator, ein Gemisch oberflächenaktiver Stoffe und ein Additiv, wobei der Emulgator Glycerinpolyethylenglycolricinoleat umfasst.
  2. Wässrige Zusammensetzung nach Anspruch 1, wobei das Gemisch oberflächenaktiver Stoffe 70 Mol-% Lecithin, 28 Mol-% Cholesterin und 2 Mol-% DPPE umfasst.
  3. Wässrige Zusammensetzung nach Anspruch 1 oder 2, wobei das Additiv Propylenglycol ist.
  4. Wässrige Zusammensetzung nach einem der Ansprüche 1 bis 3, umfassend 35,6 Gew./Vol.-% Perfluor-15-krone-5-ether, 3 Gew./Vol-% eines Emulgators, wobei der Emulgator Glycerinpolyethylenglycolricinoleat umfasst, 2 Gew./Vol.-% eines Gemischs oberflächenaktiver Stoffe, wobei das Gemisch oberflächenaktiver Stoffe Lecithin, Cholesterin und DPPE umfasst, und 2 Gew./Vol.-% eines Additivs, wobei das Additiv Propylenglycol ist.
  5. Wässrige Zusammensetzung nach einem der Ansprüche 1 bis 3, umfassend 35,6 Gew./Vol.-% PFPE-oxid, 3 Gew./Vol.-% eines Emulgators, wobei der Emulgator Glycerinpolyethylenglycolricinoleat umfasst, 2 Gew./Vol.-% eines Gemischs oberflächenaktiver Stoffe, wobei das Gemisch oberflächenaktiver Stoffe Lecithin, Cholesterin und DPPE umfasst, und 2 Gew./Vol.-% eines Additivs, wobei das Additiv Propylenglycol ist.
  6. Emulsion, umfassend eine Zusammensetzung nach einem der Ansprüche 1 bis 5.
  7. Verfahren zur Herstellung einer Emulsion nach Anspruch 6, umfassend Hoch-Energie-Verfahren.
  8. Verfahren zur Markierung einer Zelle, umfassend das in-Kontakt-bringen der Zelle ex vivo mit einer Emulsion nach Anspruch 6 unter solchen Bedingungen, dass das bildgebende Fluorkohlenstoffreagens mit der Zelle assoziiert wird, wobei die Zelle keine von einem menschlichen Embryo erhaltene Zelle ist.
  9. Verfahren zum Nachweis einer Zelle in einem Individuum, umfassend:
    a) Verabreichen einer Zelle, die mit einer Emulsion nach Anspruch 6 markiert ist, an das Individuum; und
    b) Untersuchen mindestens eines Teils des Individuums mit einer Kernspinresonanz-Technik, wodurch die markierte Zelle in dem Individuum nachgewiesen wird,
    wobei die Zelle keine von einem menschlichen Embryo erhaltene Zelle ist.
  10. Verfahren zum Messen des partiellen Sauerstoffdrucks in einem Gewebe, umfassend in-Kontakt-bringen des Gewebes in vivo mit einer Emulsion nach Anspruch 6 unter solchen Bedingungen, dass das bildgebende Fluorkohlenstoffreagens mit dem Gewebe assoziiert wird.
  11. Emulsion nach Anspruch 6 zur Verwendung in einem Verfahren zum Nachweis erhöhter vaskulärer Durchlässigkeit in einem Gewebe, wobei das Gewebe in vivo mit der Emulsion unter solchen Bedingungen in Kontakt zu bringen ist, dass das bildgebende Fluorkohlenstoffreagens mit dem Gewebe assoziiert wird.
  12. Emulsion nach Anspruch 6 zur Verwendung in einem Verfahren zum Nachweis einer Entzündung in einem Gewebe, wobei das Gewebe in vivo mit der Emulsion unter solchen Bedingungen in Kontakt zu bringen ist, dass das bildgebende Fluorkohlenstoffreagens mit dem Gewebe assoziiert wird.
  13. Markierte zelluläre Formulierung zur Verabreichung an ein Individuum, umfassend:
    a) eine Zelle; und
    b) eine Emulsion nach Anspruch 6, die mit der Zelle assoziiert ist,
    wobei die Zelle keine von einem menschlichen Embryo erhaltene Zelle ist.
EP09739238.5A 2008-05-02 2009-05-01 Zusammensetzungen und verfahren zur herstellung von emulsionen für kernmagnetresonanzverfahren und andere anwendungen Not-in-force EP2280736B1 (de)

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US9352057B2 (en) 2016-05-31
EP2280736A2 (de) 2011-02-09
WO2009134435A3 (en) 2010-02-04
US20110110863A1 (en) 2011-05-12
AU2009241762A1 (en) 2009-11-05
CA2723171A1 (en) 2009-11-05
WO2009134435A2 (en) 2009-11-05
CA2723171C (en) 2018-03-27
IL209059A0 (en) 2011-01-31
JP5751708B2 (ja) 2015-07-22
AU2009241762B2 (en) 2015-07-16
US20130343999A1 (en) 2013-12-26
ES2611103T3 (es) 2017-05-04
JP2015062693A (ja) 2015-04-09

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